Sugar-modified cytostatics

ABSTRACT

The invention relates to cytostatics which, by modification with sugar, are tumor-specific. Suitable spacers ensure serum stability and at the same time an intracellular action.

BACKGROUND OF THE INVENTION

This application is a 371 of PCT/EP96/01279, which was filed on Mar. 11,1996.

1. Field of the Invention

The invention relates to cytostatics which, due to modification withcarbohydrates, are tumour-specific. Suitable spacers ensure serumstability and at the same time an intracellular action.

2. Description of Related Art

Chemotherapy of tumour diseases is accompanied by usually serious sideeffects caused by the toxicity of chemotherapeutics on proliferatingcells of other tissue. For many years, scientists have been addressingthe problem of improving the selectivity of the active compounds used.One approach which is often followed is the synthesis of prodrugs, whichare liberated more or less selectively in the target tissue, for exampleby a change in the pH (Tietze et al., for example DE-4 229 903), byenzymes (for example glucuronidases; Jacquesy et al., EP-511 917;Bosslet et al., EP-595 133) or by antibody-enzyme conjugates (Bagshaweet al. WO 88/07378; Senter et al., U.S. Pat. No. 4,975,278; Bosslet etal., EP-595 133). Problems of these approaches are, inter alia, the lackof stability of the conjugates in other tissues and organs and, inparticular, the ubiquitous distribution of the active compound, whichfollows extracellular liberation of the active compound in the tumourtissue.

The pronounced lectin pattern on the surfaces of tumour cells (Gabius;Onkologie 12, (1989), 175) opens up the chief possibility of addressingthese specifically on tumour cells by linking the correspondingcarbohydrate units to cytostatics. These perspectives are limited by thefact that lectins with similar carbohydrate specificities (galactose,lactose, mannose, N-acetyl-glucosamine, fucose and the like) also occurin other tissues, in particular in the liver (Ashwell et al., Annu. Rev.Biochem. 46 (1982), 531; Stahl et al. Proc. Natl. Acad. Sci. U.S.A. 74(1977), 1521; Hill et al., J. Biol. Chem. 262 (1986), 7433; Jansen etal., J. Biol. Chem. 266 (1991), 3343). A significant concentration ofglycoconjugates containing the active compound in the liver and otherlectin-rich organs must consequently be expected if such non-modifiedsugars are used.

The heterocyclic amine batracyline (1) shows a good antitumoural actionin various intestinal cancer models (U.S. Pat. No. 4,757,072).

Peptide conjugates of (1) with a good in vitro action and morefavourable solubility properties (U.S. Pat. No. 4,180,343) are toleratedmore poorly than batracyline in animal studies. The fucose conjugatesdescribed in EP-501 250 become very highly concentrated in the liver.

Quinolone-a (2),7-[(3aRS,4RS,7aSR)-4-amino-1,3,3a,4,7,7a-hexahydro-isoindol-2-yl]-8-chloro1-cyclopropyl-6-fluoro-1,4-dihydro4-oxo-3-quinolinecarboxylicacid also shows, in addition to its outstanding antibacterial activity,a very good activity against various tumour cell lines (EP-520 240, JP-4253 973). However, this is faced with considerable toxicologicalproblems (for example genotoxicity, bone marrow toxicity, high acutetoxicity in vivo and the like).

BRIEF SUMMARY OF THE INVENTION

By a novel modification of cytostatics, we have found, surprisingly, anew class of conjugates which are distinguished by the followingproperties: A novel linkage of carbohydrates with cytostatics (forexample batracyline, quinolone-a) leads to glycoconjungates which areserum-stable. The action does not depend on extracellular liberation ofthe active compound. The in vitro activities against various tumour celllines are comparable to that of the cytostatic on which they are based.The cell-specific absorption depends on the carbohydrate.

The cell and tissue selectivity (in particular tumour to liver) issignificantly improved by the regioselective modifications in thecarbohydrate part of the conjugates described.

In vivo, the conjugates according to the invention are distinguished bya significantly improved tolerance compared to the active compound andthe corresponding peptide conjugates.

Furthermore, in comparison with the cytostatics on which they are based,the conjugates according to the invention show considerably improvedsolubility properties.

DETAILED DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing lack of inhibition of bone marrow parent cellproliferation.

DETAILED DESCRIPTION OF THE INVENTION

The compounds according to the invention are described by the followinggeneral formula:

K-Sp-L-AA1-AA2-C  (I)

where

K=an unsubstituted or regioselectively modified carbohydrate radical,

Sp=optionally substituted arylene or alkylene,

where

R=chlorine or hydroxyalkylamino, the linkage to Sp being via the NHgroup.

AA1 is an amino acid radical in the D or L configuration, whichoptionally carries a second grouping K-Sp-L-, in which K, Sp and L,independently of the other grouping K-Sp-L-, can have the abovementionedmeanings, or a direct bond. An amino acid radical can be linked to Lboth via the α-amino group and, where appropriate, via side chain aminoor hydroxyl functions, and also via both functions. If AA1 carriesfurther functional groups, these can be present in deblocked form or ina form protected with known protective groups. Suitable protectivegroups are, for example, acetyl, allyloxycarbonyl, benzyloxycarbonyl,fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl ortert-butyl.

AA2 is an amino acid radical in the D or L configuration, whichoptionally carries a second grouping K-Sp-L-, in which K, Sp and L,independently of the other grouping K-Sp-L-, can have the abovementionedmeanings, or a direct bond. An amino acid radical can be linked to AA1both via the α-amino group and, where appropriate, via side chain aminoor hydroxyl functions, and also via both functions. If AA2 carriesfurther functional groups, these can be present in deblocked form or ina form protected with known protective groups. Suitable protectivegroups are, for example, benzyloxycarbonyl, acetyl, allyloxycarbonyl,fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl ortert-butyl.

C=radicals of a cytostatic or of a cytostatic derivative, which canadditionally carry an amino or hydroxyl group. C can be an intercalatingsubstance, a topoisomerase inhibitor, an antimetabolite, an alkylatingagent, a tubulin inhibitor, a tyrosine phosphokinase inhibitor, aprotein kinase-C-inhibitor or an active compound with another or anunknown cytostatic or cytotoxic action mechanism. C can be, for example,a nucleoside, an endiine antibiotic, a quinolone- ornaphthyridone-carboxylic acid or a cytotoxic peptide antibiotic, forexample from the class of dolastatins. C can be batracyline,quinolone-a, 5-fluorouracil, cytosine arabinoside, methotrexate,etoposide, camptothecin, daunomycin, doxorubicin, taxol, vinblastine,vincristine, dynemycin, calicheamycin, esperamycin, quercetin, suramin,erbstatin, cyclophosphamide, mitamycin C, melphalan, cisplatin,bleomycin, staurosporin or another active compound having anantineoplastic action.

The structural element -Sp-L-AA1-AA2- in total represents the spacerwhich connects K and C.

Preferred compounds of the formula (I) are those in which

K=a carbohydrate radical having the general formula

wherein

A=methyl, hydroxymethyl, carboxyl and esters and amides derivedtherefrom, alkoxymethyl, acyloxymethyl or carboxyalkyloxymethyl andesters and amides derived therefrom. A can also be CH₂—B, wherein B inturn can be a carbohydrate radical of the general formula (II) linkedvia the anomeric centre.

R₂, R₃, R₄=individually or together at the same time, H, hydroxyl,alkyloxy, carboxyalkyloxy and esters and amides derived therefrom,hydroxyalkyloxy, aminoalkyloxy, acyloxy, carboxyalkylcarbonyloxy,sulphato, phosphate, halogen or another carbohydrate radical (II)modified in the same framework and linked via the anomeric centre. R₂can additionally also be amino or acylamino.

Two of the radicals R₂, R₃ or R₄ together can also denote an epoxidegroup.

The stereochemistry on the anomeric centre of the carbohydratestructural unit can be α or β. The gluco, manno, galacto, gulo, rhamnoor fuco configuration can result from the stereochemistry on the othercentres.

Sp=an arylene radical which is modified with K and L in the ortho-,meta- or para-position and furthermore can also carry 1 to 4 furthersubstituents which, independently of one another or in an identicalmanner, can be H, methyl, methoxy, hydroxyl, carboxyl, methoxycarbonyl,cyano, nitro, halogen, sulphonyl or sulphonamide;

Sp can also be a linear or branched alkylene radical.

where

R=chlorine or hydroxyalkylamino,

AA1 is an amino acid radical in the D or L configuration, whichoptionally carries a second grouping K-Sp-L-, in which K, Sp and L,independently of the other grouping K-Sp-L-, can have the abovementionedmeanings, or a direct bond. An amino acid radical can be linked with Lboth via the α-amino group and, where appropriate, via side chain aminoor hydroxyl functions and also via both functions. If AA1 carriesfurther functional groups, these can be present in deblocked form or ina form protected with known protective groups. Suitable protectivegroups are, for example, acetyl, allyloxycarbonyl, benzyloxycarbonyl,fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl ortert-butyl.

AA2 is an amino acid radical in the D or L configuration, whichoptionally carries a second grouping K-Sp-L-, in which K, Sp and L,independently of the other grouping K-Sp-L-, can have the abovementionedmeanings, or a direct bond. An amino acid radical can be linked with AA1both via the α-amino group and, where appropriate, via side chain aminoor hydroxyl functions, and also via both functions. If AA2 carriesfurther functional groups, these can be present in deblocked form or ina form protected with known protective groups. Suitable protectivegroups are, for example, benzyloxycarbonyl, allyloxycarbonyl, acetyl,fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl ortert-butyl.

C can be, for example, the radical of a nucleoside, an endiineantibiotic or a cytotoxic peptide antibiotic, for example from the classof dolastatins, or a quinolone- or naphthyridonecarboxylic acid asdefined below. C can be, for example, batracyline, 5-fluorouracil,cytosine arabinoside, methotrexate, etoposide, camptothecin, daunomycin,doxorubicin, taxol, vinblastine, vincristine, dynemycin, calicheamycin,esperamycin, quercetin, suramin, erbstatin, cyclophosphamide, mitamycinC, melphalan, cisplatin, bleomycin, staurosporin or another activecompound having an antineoplastic action. The cytostatic is linked withAA2 via amino or hydroxyl functions.

Especially preferred compounds of the formula (I) are those in which

K=a carbohydrate radical of the general formula

wherein

A=methyl, hydroxymethyl, carboxyl and methoxycarbonylmethyl and CH₂—B,wherein B in turn can be a carbohydrate radical of the general formula(II) linked via the anomeric centre.

R₂, R₃ and R₄=individually or together at the same time, H, hydroxyl,C₁-C₃-alkyloxy, carboxy-C₁-C₃-alkyloxy and C₁-C₃-alkyl esters and amidesderived therefrom, hydroxyalkyloxy, acyloxy,carboxy-(C₁-C₃-alkyl)-carbonyloxy, sulphato or another carbohydrateradical in position R₃ or R₄ linked via the anomeric centre.

Two of the radicals R₂, R₃ and R₄ together can also denote an epoxidegroup.

The stereochemistry on the anomeric centre can be α or β. The D-manno,D-galacto, L-gulo, D-gluco, L-rhamno or L-fuco configuration can resultfrom the stereochemistry on the other centres.

Sp=an arylene radical which is modified with K and L in the ortho- orpara-position and furthermore can also carry, in addition to hydrogenatoms, a further substituent, which can be methoxy, nitro or chlorine;

where

R=chlorine or hydroxyalkylamino.

AA1 is an amino acid radical, such as lysine, alanine, aspartic acid,glutamic acid, glycine, ornithine, tyrosine, valine or serine in the Dor L configuration, or a direct bond. The amino acid radical can belinked with L both via the α-amino group and, where appropriate, via theside chain amino functions, and also via both functions, and thusoptionally carries a further grouping K-Sp-L-, which is identical to ordifferent from the first. If AA1 carries further functional groups,these are preferably deblocked.

AA2 is an amino acid radical, such as alanine, lysine, glycine, serine,ornithine or diaminopropionic acid in the D or L configuration, or adirect bond. The amino acid radical can be linked with AA1 both via theα-amino group and, where appropriate, via the side chain aminofunctions, and also via both functions, and can thus optionally carry afurther grouping K-Sp-L-, which is identical to or different from thefirst. If AA2 carries further functional groups, these are preferablydeblocked.

C can be batracyline, methotrexate, quinolone-a, etoposide, melphalan,taxol, camptothecin, daunomycin or doxorubicin or a quinolone- ornaphthyridonecarboxylic acid as defined below. The cytostatic is linkedwith AA2 via an amino or hydroxyl function.

The quinolone- or naphthyridonecarboxylic acid structural units C usedas educts can be represented by the general structure of the formula(III)

T-Q  (III)

in which

Q denotes a radical of the formulae

wherein

R^(a) represents alkyl which has 1 to 4 carbon atoms and is optionallymono- or disubstituted by halogen or hydroxyl, vinyl, cycloalkyl whichhas 3 to 6 carbon atoms and is optionally substituted by 1 or 2 fluorineatoms, bicyclo[1.1.1]pent-1-yl, 1,1-dimethylpropargyl, 3-oxetanyl,methoxy, amino, methylamino, dimethylamino or phenyl which is optionallymono- or disubstituted by halogen, amino or hydroxyl, or, together withR^(e), can also form a bridge described for that radical,

R^(b) represents hydroxyl, alkoxy having 1 to 3 carbon atoms ornitromethyl,

R^(c) represents hydrogen or methyl or, together with R^(g), can alsoform a bridge described for that radical,

R^(d) represents hydrogen, CH₃, CH₂F or ═CH₂,

X¹ represents hydrogen, halogen or nitro,

X² represents hydrogen, halogen, amino, hydroxyl, methoxy, mercapto,methyl, halogenomethyl or vinyl,

Y represents N or C—R^(e), wherein

R^(e) represents hydrogen, halogen, CF₃, OCH₃, OCHF₂, CH₃, CN, CH═CH₂ orC═CH, or, together with R^(a), can also form a bridge of the structure—*O—CH₂—CH—CH₃, —*S—CH₂—CH₂—, —*S—CH₂—CH—CH₃, —*CH₂—CH₂—CH-CH₃ or—*O—CH₂—N—R^(f), wherein the atom marked with is linked with the carbonatom of Y and wherein

R^(f) denotes hydrogen, methyl or formyl, and

D represents N or C—R^(g), wherein

R^(g) represents hydrogen, halogen, CF₃, OCH₃, OCHF₂ or CH₃, or,together with R^(c), can also form a bridge of the structure —*O—CH₂—,—*NH—CH₂—, —*N(CH₃)—CH₂—, —*N(C₂H₅)—CH₂—, —*N(C₃H₅)—CH₂— or —*S—CH₂—,wherein the atom marked with * is linked with the carbon atom of D,

n denotes 1, 2 or 3 and

T denotes a radical of the formula

wherein

R^(h) represents

wherein

R^(k) represents hydrogen or methyl and

R^(i) represents hydrogen, C₁-C₃-alkyl or cyclopropyl.

Compounds of the formula (III) which are particularly preferred as thecytostatic C are those in which

Q denotes a radical of the formula

wherein

R^(a) represents alkyl which has 2 to 4 carbon atoms and is optionallysubstituted by 1 fluorine atom, cyclopropyl which is optionallysubstituted by 1 fluorine atom or phenyl which is optionally mono- ordisubstituted by fluorine,

R^(b) represents hydroxyl or alkoxy having 1 or 2 carbon atoms,

R^(c) represents hydrogen or methyl, or, together with R^(g), can form abridge described for that radical,

X¹ represents fluorine,

X² represents hydrogen or amino,

Y represents N or C—R^(e), wherein

R^(e) represents hydrogen, fluorine, chlorine, CF₃, OCH₃, OCHF₂ or C≡CH,or, together with R^(a), can form a bridge of the structure—*O—CH₂—CH—CH₃ or —*O—CH₂—N—R^(f), wherein the atom marked with * islinked with the carbon atom of Y and wherein R^(f) denotes methyl,

D represents N or C—R^(g), wherein

R^(g) represents hydrogen, fluorine, chlorine, CF₃, OCH₃ or CH₃, or,together with R^(e), also form a bridge of the structure —*O—CH₂—,—*N—CH₂—, —N(CH₃)—CH₂— or —*S—CH₂—, wherein the atom marked with islinked with the carbon atom of D, and

T denotes a radical of the formula

 wherein

R^(h) represents

 wherein

R^(k) represents hydrogen or methyl, and

R^(i) represents hydrogen or methyl.

Glycoconjugates with camptothecin or derivatives thereof are likewiseparticularly preferred.

The compounds of the general formula I in which K, Sp and L denotehydrogen and

C represents camptothecin are furthermore of particular importance.These substances are new and can be reacted as intermediate products togive further derivatives of the general formula I, and in turn also showan interesting pharmaceutical action spectrum, in particular ascytostatics.

The compounds according to the invention can be in stereoisomeric forms,for example as enantiomers or diastereomers, or in the form of mixturesthereof, for example as a racemate. The invention relates both to thepure stereoisomers and to mixtures thereof.

If necessary, the stereoisomer mixtures can be separated into thestereoisomerically uniform constituents in a known manner, for example,by chromatography or by crystallization processes.

The compounds according to the invention can also be in the form oftheir salts. Salts with organic or inorganic bases or acids and innersalts may be mentioned in general here.

The acids which can be added on include, preferably, hydrogen halideacids, such as, for example, hydrochloric acid and hydrobromic acid, inparticular hydrochloric acid, and furthermore phosphoric acid, nitricacid, sulphuric acid, mono- and bifunctional carboxylic acids andhydroxycarboxylic acids, such as, for example, acetic acid, maleic acid,malonic acid, oxalic acid, gluconic acid, succinic acid, fumaric acid,tartaric acid, citric acid, salicylic acid, sorbic acid and lactic acid,and sulphonic acids, such as, for example, p-toluenesulphonic acid,1,5-naphthalene-disulphonic acid or camphorsulphonic acid.

Physiologically acceptable salts can likewise be metal or ammonium saltsof the compounds according to the invention which have a free carboxylgroup. Particularly preferred salts are, for example, sodium, potassium,magnesium or calcium salts, and ammonium salts which are derived fromammonia or organic amines, such as, for example, ethylamine, di- ortriethylamine, di- or triethanolamine, dicyclohexylamine,dimethylaminoethanol, arginine, lysine, ethylenediamine orphenethylamine.

EXAMPLE SERIES A Biological Testing EXAMPLE A.1

Growth Inhibition Test for Determination of the Cytotoxic Properties ofGlycoconjugates of Batracyline and of Quinolone-a

The human colon cell lines SW 480 and HT 29 (ATCC no. CCL 228 andHBT-38) and the mouse melanoma cell line B 16 F 10 were cultured in Rouxdishes in RPMI 1640 medium with addition of 10% FCS. The cultures werethen trypsinized and taken up in RPMI plus 10% FCS to a cell count of50,000 cells/ml. 100 μl of cell suspension/well were introduced into a96 microwell plate and incubated for 1 day at 37° C. in a CO₂ incubatingcabinet. A further 100 μl of RPMI medium and 1 μl of dimethyl sulphoxidewith the test substances were then added. The growth was checked afterday 3 and day 6. 40 μl of MTT solution(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoline bromide) with aninitial concentration of 5 mg/ml of H₂O were added to each microwave.The plate was incubated for 5 hours in a CO₂ incubating cabinet at 37°C. The medium was then sucked off and 100 μl of i-propanol/well wereadded. After shaking for 30 minutes with 100 μl of H₂O, the extinctionwas measured at 540 nm with a Titertek Multiskan MCC/340 (Flow).

The cytotoxic action of the glycoconjugates of batracyline described isshown in Table 1a as the IC₅₀ value in each case for the SW 480 and HT29 cell lines.

The IC₅₀ values for the quinolone-a glycoconjugates on the SW 480, HT 29and B 16 F 10 cell lines are summarized in Table 1b.

TABLE 1a IC₅₀ [μM] IC₅₀[μM] Substance SW 480 HT 29 Batracyline 25 203.2  100 75 3.4  100 65 3.7  55 n.m  3.9  40 55 3.10 100 125  3.11 85n.m. 3.14 40 n.m. 3.18 15 n.m. 3.19 75 n.m. 3.20 100 n.m. 3.21 90 n.m.3.23 50 n.m. 3.24 95 n.m. 3.26 50 n.m. 3.27 110 n.m. 3.28 60 n.m. 3.29110 n.m. 3.30 >250 n.m. 3.33 90 70 4.1  25 30 4.3  20 20 4.4  30 25 4.5 15 15 4.6  10 10 4.7  15 15 4.8  50 40 4.9  20 30 4.10 30 30 4.11 15 154.12 15  9 5.1  55 45 5.2  20 55 5.6  >250 n.m. 5.8  100 >250    5.9  7070 5.12 20 20 5.13 20 40 5.14 20 30 5.15 >250 70 5.19 35 25 5.20 50 305.21 60 80 5.22 30 40 5.23 25 35 6.2  40 50 6.3  70 105  6.7  60 >250   6.10 50 50 6.12 35 50 6.15 >250 >250    6.20 80 n.m. 6.21 150 n.m. 6.23107 45 6.25 50 40 6.28 40 25 6.29 95 130  6.30 60 70 6.32 60 60 6.34 50n.m. 6.35 20 n.m. 6.36 70 70 6.40 170 60 6.43 90 80 6.46 120 100  6.5950 50 6.60 50 40 6.80 40 25 6.81 30 30 6.82 125 n.m. 6.83 90 n.m. 6.8522 n.m. 7.1  40 40 7.2  40 30 7.3  50 n.m. 7.5  80 n.m. 7.7  100 >250   7.8  40 30 7.11 30 25 7.12 10 10 8.10 70 n.m. 8.11 45 n.m. 8.12 30 n.m.

TABLE 1b IC₅₀[μM] IC₅₀[μM] IC₅₀[μM] Substance SW 480 HT 29 B 16 F 1010.1 50 >250 15 10.2 4 3 5 10.3 5 4 0.7 11.2 30 n.m. 9 11.6 8 9 5 11.712 13 15 11.8 12 16 15 11.9 12 12 9 11.10 8 20 2 11.16 10 10 1.5 11.1775 75 8 11.18 4.5 3.5 0.5 12.1 1 1.5 0.1 12.2 4 n.d. 0.8 12.3 2 n.d. 0.312.5 1 4 0.2 12.6 4 7 0.3 12.7 60 >250 20 12.8 8 7 1 12.9 4 8 2 12.10 1515 4 12.11 2 2 0.5 12.12 8 13 0.5 12.13 35 100 1 12.14 1 2 0.3 12.15 0.31 0.1 14.1 0.8 1 1.5 14.2 1 6 1.5 14.3 8 4 4 14.4 1.5 1 0.4 15.1 20 20 215.2 50 70 15 16.1 50 100 200 16.2 50 60 80 17.1 10 5 5 17.2 4 4 4 18.10.03 0.01 0.2 18.2 0.02 0.02 0.2 18.4 0.02 0.02 0.3 18.5 0.2 0.2 1 18.90.08 0.06 0.7 18.14 0.015 0.01 0.08

The dependence of the biological action on the carbohydrate isadditionally demonstrated by the inactivity of the carbohydrate-freecomparison compoundsN-[N^(α),N^(ε)-bis-(4-hydroxyphenylamino-thiocarbonyl)-lysyl]-batracylineandN-[N^(α),N^(ε)-bis-(4-hydroxyphenylamino-thiocarbonyl)-lysyl-D-alanyl]-batracylineandN-[N^(α),N^(ε)-bis-(4-hydroxyphenylamino-thiocarbonyl)-D-lysyl-quinolone-a(IC₅₀ values>250), on which Example series 5, 6 and 11 are based.

EXAMPLE A.2

In Vitro Investigation of the Cleavability of the Glycoconjugates

Cleavage Kinetics With Human Blood

1.225 ml of human blood are incubated together with 1.25 ml of PBS and25 μl of a substrate stock solution (1 mg/ml in 3% dimethyl sulphoxidein PBS) at 37° C. After 1 hour and 24 hours, samples of in each case 1ml are taken, mixed with 1 ml of ethanol and left to stand at 4° C. for20 minutes. After centrifugation (5 minutes at 3500 rpm), 100 μl ofsupernatant are taken for the HPLC analysis.

Cleavage Kinetics With Cells

2.25 ml of PBS are incubated together with 225 μl of a cell suspension(30 mg/ml) and 25 μl of substrate stock solution (1 mg/ml in 3% dimethylsulphoxide in PBS) at 37° C. After 1 hour and 24 hours, samples of ineach case 1 ml are taken, mixed with 1 ml of ethanol and left to standat 4° C. for 20 minutes. After centrifugation (5 minutes at 3500 rpm),100 μl of supernatant are taken for the HPLC analysis.

HPLC Conditions

Apparatus:

Waters unit

Column:

Bischoff Hypersil OCS RP 18 5 μm 250×4 mm

Eluent:

A: 10 mM potassium phosphate buffer, pH 4.5

B: 80% acetonitrile/20% water

Flow:

1 ml/minute

Wavelength:

372 nm

Gradient:

0 minute 10% B

10 minutes 60% B

15 minutes 60% B

18 minutes 10% B

20 minutes 60% B

Eluent for Quinolone-a Conjugates

A: 100% methanol

B: 10 mM potassium phosphate buffer, pH 2.2;

10 mM heptanesulphonic acid

TABLE 2a % cleavage in % cleavage in % cleavage in Exam- human bloodSW-480 hepatoma ple 1 hour 24 hours 1 hour 24 hours 1 hour 24 hours 3.4n.d. n.d. 74* n.d. 98* n.d. 3.9 0 54* 28* 100*  32* 100*  4.4 0 0 0 0 00 5.9 0 0 0 0 0 0 6.2 n.d. n.d. 0 0 0 0 6.12 n.d. n.d. 0 0 0 0 7.3 0 0 00 0 0 *Cleavage product is N-[D-alanyl]-batracyline

TABLE 2b % cleavage in % cleavage in % cleavage in Exam- human bloodSW-480 hepatoma ple 1 hour 24 hours 1 hour 24 hours 1 hour 24 hours 11.20 0 0 0 0 0 11.6 0  11% 0    8% 0  12% 11.7 0 0 0 0 0 0 12.1 0 0 0 0 0 012.3 0 0 0 0 0 0 12.8 0 0 0 0 0 0

EXAMPLE A.3

Investigation of Organ Distribution

Athymic naked mice (strain NMRI nu/nu) bred in “Drug DevelopmentLaboratory, Oncotest GmbH”, Prof. H. H. Fiebig, Freiburg, were used forall the experiments. The animals were kept in Macrolon cages underlaminar flow conditions. Tissue of the cell line SW 480 which had beengrown beforehand in several passages in the naked mice, was used as thetumour material.

Two tumours per animal were implanted subcutaneously in the two flanksof the naked mice 6 to 8 weeks old. The animals were kept for 26 to 27days until the time of randomization. The average tumour size was then500 mg, corresponding to a tumour diameter of about 10 mm.

The pharmacokinetics themselves proceeded as follows: the substance tobe investigated was injected into the naked mice and the mice were thenput back in the cages until samples were taken after ½ hour and after 4hours. The taking of samples itself began with sampling of blood. Forthis, the mouse was anaesthetized by means of anaesthetic ether(duration ½ to one minute). 0.5 hour and 4 hours after injection of thesubstance, the abdominal cavity was opened and the mouse wasexsanguinated under anaesthesia via the Vena cava caudalis in the courseof 1 to 2 minutes and then sacrificed by breaking its neck. As a result,central circulatory arrest occurred and organ perfusion was suppressed.The individual organs were then exposed and removed, an operation whichtook about 5 minutes. Immediately thereafter, the organ samples and thenthe remainder of the body were weighed and frozen in liquid nitrogen.

The substance “conjugate 1” is administered i.p. in an amount of 300mg/kg of body weight and the substance “conjugate 2” is administeredi.v. into the tail vein in an amount of 100 mg/kg. 5 animals are usedper substance and time.

The distribution results are summarized in Table 3 for conjugate 1 andin Table 4 for conjugate 2.

A. Calibration Series

5, 10, 50, 100 and 200 μg of substance, dissolved in ethanol/water (1:1,v/v) were added to 1 g of bovine liver. The samples were then ground ina mortar with 1 g of sea sand and 2.5 ml of cooled ethanol/water (1:1,v/v) and centrifuged at 3500 rpm for 2 minutes. After removal of thesupernatant, the residue was again mixed with 2.5 ml of ethanol/waterand centrifuged and the supernatants were combined. In each case 100 μlwere taken and analyzed by HPLC.

HPLC Conditions

Apparatus:

Waters unit

Column:

Bischoff Hypersil OCS RP 18 5 μm 250×4 mm

Eluent:

A: 80% acetonitrile/20% water

B: 10 mM potassium phosphate buffer, pH 4.5

Gradient:

0 minute 90% B

10 minutes 40% B

15 minutes 40% B

18 minutes 90% B

20 minutes 90% B

Flow:

1 ml/minute

Wavelength:

372 nm

B. Working up of the Organs

The organs were worked up analogously to A, the entire organs beingbroken down with 2.5 ml of ethanol/water and extracted.

C. Working up of the Blood

The amount of blood taken was mixed with 2 ml of ethanol/water (1:1,v/v) and centrifuged for 2 minutes at 3500 rpm, and the supernatant wasdecanted. 2 ml of ethanol/water (1:1, v/v) were again added to theresidue, the mixture was centrifuged and the supernatants were combined.In each case 100 μl of the combined supernatants were analyzed by HPLC.The HPLC conditions used under A were used.

TABLE 3 Evaluation of organ samples Mean 1 hour Mean 4 hours OrganSubstance (μg/g of organ) (μg/g of organ) Blood Conjugate 1 31.2 —Batracyline — — Tumour Conjugate 1 56.6 24.4 Batracyline — — LiverConjugate 1 770   126   Batracyline 34.1 22.4 Kidney Conjugate 1 81  78.4 Batracyline 26.1 27.5 Brain Conjugate 1 — — Batracyline — —

TABLE 4 Evaluation of organ samples Mean 0.5 hour Mean 4 hours OrganSubstance (μg/g of organ) (μg/g of organ) Blood Conjugate 2 6.5 —Batracyline — — Tumour Conjugate 2 2.0 — Batracyline 2.5  3.12 LiverConjugate 2 0.9 1.2 Batracyline — — Kidney Conjugate 2 17.5  0.5Batracyline 10.7  0.8 Brain Conjugate 2 — — Batracyline — —

EXAMPLE A.4

Determination of the Acute Toxicity of Glycoconjugates of Batracyline(Single Administration)

The acute toxicity of the batracyline derivatives was determined onnu/nu naked mice.

The substances administered i.v., as aqueous solutions, and thesubstances administered i.p., as solutions in dimethyl sulphoxide, wereinjected once in concentrations of up to 400 mg of substance per kg ofmouse.

The individual dose tolerated was calculated from the decrease in theweight of the animals up to 21 days after administration and from thenumber of animals surviving.

The individual dose of the substances which can be tolerated can be seenfrom Table 5.

For substances 3.16; 3.33; 3.9; 6.12; 6.14; 6.2; 6.81; and 8.2 it wasmore than 200 mg of substance per kg of mouse. For substances 3.33;6.12; 6.14; 6.2; and 6.81, still no acute toxicity was to be detectedeven with a dose of 400 mg/kg.

In contrast, sugar-free lysyl-D-alanyl-batracyline (2.13) was alreadysignificantly toxic at individual doses of between 25 and 50 mg/kg ofmouse.

TABLE 5 Maximum individual dose of batracyline derivatives andquinolone-a derivatives which can be tolerated Individual dose toleratedin mg/kg of mouse Example i.p. i.v. 2.13    50    25 3.4    200 <100 (2animals died) 3.9    200   200 3.16 >200 n.d. 3.33 n.m. >400 6.2  >400n.m. 6.12 n.m. >400 6.14 >400 n.m. 6.81 n.m. >400 8.2  >200 n.d.Quinolone-a n.d.  <25 12.3  n.d. >200

EXAMPLE A.5

Determination of the Acute Toxicity After Several Administrations

The substances were administered partly i.v. and partly i.p., eitherdaily on days 1 to 4 and 7 to 10 or on days 1, 5 and 9. The dosages were400, 200 and 100 mg/kg/day. The evaluation was made according to thedecrease in weight up to day 21 and according to the number of survivinganimals. For the experiments, 5 animals were employed per substance anddose. The results are summarized in Table 6.

TABLE 6 Maximum tolerable dose with multiple administrationsAdministration Dosage on day MTD mg/kg day 3.9  i.v. 1-4, 7-10  ˜50 1-4˜100 3.33 i.v. 1, 5, 9 >400 6.2  i.p. 1-4, 7-10 ˜400 1, 5, 9 >400 6.12i.v. 1-4, 7-10 >400 1, 5, 9 >400 6.14 i.p. 1, 5, 9 >400 6.81 i.v. 1-4,7-10 ˜200 >400 Batracyline i.p 1, 5, 9 −100

EXAMPLE A.6

Haematopoietic Activity of Glycoconjugates of Quinolone-a in ComparisonWith the Active Compound on Which They Are Based

Material and Methods

In vitro:

Bone marrow cells are flushed out of the femur of mice. 10⁵ cells areincubated in McCoy SA medium (0.3% agar) together with recombinantmurine GM-CSF (Genzyme; parent cell colony formation) and the substances(10⁻⁴ to 100 μg/ml) at 37° C. and 7% CO₂. 7 days later, the colonies(<50 cells) and clusters (17-50 cells) are counted.

In vivo:

Mice are treated subcutaneously with 1, 3, 10 or 30 mg/kg of thecompounds. At various times (3, 24, 48, 72 p. inj.) the femurs areremoved and the bone marrow cells isolated. 2×10⁵ cells are incubatedwith GM-CSF as described above and, after 7 days, the colonies andclusters are counted.

Results

As shown in Table 7, the glycoconjugates investigated show an inhibitionof bone marrow parent cell proliferation which is reduced by a factor of10⁵ to 10³ compared with quinolone-a.

In vivo also, no inhibition in parent cell proliferation was to beobserved by compound 12.3 up to 30 mg/kg, compared with quinolone-a. Amassive suppression of parent cell proliferation was already inducedwith 3 mg/kg of quinolone-a (FIG. 1).

TABLE 7 CSF-induced proliferation of bone marrow parent cells of themouse Examples IC₅₀ [μg/ml] Quinolone-a 0.0002 11.2 22.5 11.7 2.9 12.10.21 12.3 0.27 12.6 0.3 12.8 0.3 14.1 2.9 14.2 3.6 14.4 3.6

EXAMPLE A.7

Antineoplastic Activity of Quinolone-a Conjugates

The in vitro activity of glycoconjugates of quinolone-a was determinedon human tumour xenografts in a two-layer soft agar culture systemaccording to Hamburger and Salmon (Science 197: 461-463).

The solid tumours were initially grown in the athymic naked mouse (NMRInu/nu), obtained by surgery and comminuted mechanically. Individualcells were then obtained by incubation in an enzyme mixture ofcollagenase 0.05%, DNAse 0.07% and hyaluronidase 0.1% in RPMI at 37° C.for 30 minutes. The cells were washed 2× and then passed through a sieveof 200 μm and 20 μm mesh width.

The following culture method was used:

The base layer comprises 0.2 ml of Iscoves's Modified Dulbeccos Mediumwith 20% foetal calf serum and 0.7% agar. 40,000 to 200,000 cells in 0.2ml of the same medium and 0.4% agar were applied to this layer in 24multiwell plates. The cytostatic substances were added in 0.2 ml ofmedium.

The cultures were incubated at 37° C. in a 7% CO₂ atmosphere for 6 to 15days. The colonies which had grown were then counted under an invertingmicroscope, the living colonies being stained with a tetrazoliumchloride dyestuff 24 hours before the evaluation.

The effect of the active compound is expressed in per cent of survivingcolonies compared with the colony count of untreated plates (T/C=colonycount treated×100/control count untreated).

A substance is active if the T/C value is ≦30%.

This value is shown as the IC₇₀ value in μg/ml in Table 8.

TABLE 8 IC₇₀/μg/ml Example 11.7 12.1 12.3 12.5 Quinolone-a CXF280 >100    70 5 <0.3 6 HT 29 56 6 5 11 20 SW 480 18 8 11 10 3 LXFL 529 4 0.9 2 2 3 LXFS 538 18 0.4 0.5 <0.3 <0.3 MEXF 989  3 <0.3 <0.3 0.5<0.3 OVXF 899 234  45 162 55 2 OVXF 1023 136  12 10 9 0.5

EXAMPLE A.8

In Vivo Tests

Method

Mice are inoculated with 5×10⁶ B16F10 tumour cells on day 0. The animalsin which tumour cells are transplanted develop solid peritoneal tumoursand are then treated daily with test substances or with the vehiclecontrol. In the control group, 50% of the animals usually die betweenday 14 and 20. The test substances are administered in buffer or anorganic solvent system comprising 20% methanol and 20% dimethylsulphoxide in 0.7% sodium chloride solution.

The administration of the vehicle showed no influence on the survivaltime of the animals. The therapeutic result arises from the prolongingof the survival time of the treated animals. The tolerance of thecompounds is analysed in parallel on animals which do not carry tumours.A therapeutic index can be estimated from the tolerance and prolongingof the survival time.

TABLE 9 % survivors Day 0 Day 20 Day 25 Day 30 Day 35 Control 100 70 3010 10 11.12 1 mg/kg 100 90 60 30 30 11.12 (100 mg/kg) 100 100  100  9090 Quinolone-a 100 100  100  60 40 (0.1 mg/kg) Etoposide 100 90 80 80 70(5 mg/kg)

Table 9 shows the therapeutic activity of the compound from Example11.12 on mice in which a B16F10 tumour was transplanted.

EXAMPLE A.9

In Vivo Inhibition of Tumour Growth Using a Naked Mouse Model

Material

Athymic naked mice (NMRI nu/nu strain) were used for all the in vivoexperiments for investigation of the inhibition of tumour growth. Thelarge-cell lung carcinoma LXFL 529 selected was developed by serialpassage in naked mice. The human origin of the tumour was demonstratedby isoenzymatic and immunohistochemical methods.

Experimental Set-up

The tumour was implanted subcutaneously in both flanks of nu/nu nakedmice 6 to 8 weeks old. Treatment was started, regardless of the doublingtime, as soon as the tumours had reached a diameter of 5-7 mm. The micewere allocated to the treatment group and the control group (5 mice pergroup with 8-10 evaluable tumours) by randomization. The individualtumours of the control group all grew progressively.

The size of the tumours was measured in two dimensions by means of asliding gauge. The tumour volume, which correlates well with the cellcount, was then used for all the evaluations. The volume was calculatedaccording to the equation “length×breadth×breadth/2” ([a×b²]/2, a and brepresent two diameters at right angles). The values of the relativetumour volume (RTV) were calculated for each individual tumour bydividing the tumour size on day X with the tumour size on day 0 (at thetime of randomization). The mean RTV values were then used for thefurther evaluation.

The inhibition of the increase in the tumour volume (tumor volume of thetest group/control group, T/C, in per cent) was the concludingmeasurement value.

Treatment

All the compounds were administered according to an intermittent plan ineach case on day 1, 5 and 9. Furthermore, all the compounds wereadministered intraperitoneally (i.p.) using water as the solvent.

TABLE 10 Dose^(a)) Survival time Number of Relative tumour volumeOptimum Treatment [mg/kg/day] (days) tumours [% of day 0]^(b)) T/C^(b))Control —   19 >21 10  1552  100% group >21 >21   14 12.6 100    23 >268 300.7 19.4%   26 >26 >26 12.8 50 >21 >21 9 502.2 32.3% >21 >21 >2112.14 25   23 >26   19   26 8 519.5 33.5% >26 a) maximum tolerated dose(MTD) b) on day 19

In this test, the camptohecin compounds of Example series 18 as a ruleshowed a comparable or better action.

EXAMPLES SERIES B Synthesis Examples EXAMPLE 1.1

p-Aminophenyl 2-O-methyl-β-L-fucoside

1.1.a) p-Nitrophenyl 3,4-O-isopropylidene-β-L-fucoside

65 mg of p-toluenesulphonic acid and, at 30 minute intervals, 5×100 μlof 2-methoxypropene are added to a solution of p-nitrophenylβ-L-fucoside (750 mg, 2.63 mmol) in 40 ml of dimethylformamide/dioxane1:2 at 0° C. After the mixture has been stirred at 20° C. for 16 hours,it is concentrated and the residue is purified by flash chromatography(methylene chloride/methanol 99:1). After concentration, 710 mg (83%) ofa white solid are obtained.

1.1.b) p-Nitrophenyl 2-O-methyl-3,4-O-isopropylidene-β-L-fucoside

100 mg (0.307 mmol) of the compound from Example 1.1.a are initiallyintroduced into 10 ml of tetrahydrofuran together with 96 μl of methyliodide, and 11 mg of sodium hydride (80% strength) are then added inportions. After the mixture has been stirred at 20° C. for 3 hours, itis topped up with a further 96 μl of methyl iodide and 11 mg of sodiumhydride. After further stirring at 20° C. for 16 hours, a little waterand 100 ml of methylene chloride are added. The batch is extracted byshaking twice with water, the organic phase is concentrated and theproduct is then purified by column chromatography (petroleum ether/ethylacetate 8:1). Yield: 78 mg (75%).

1.1) p-Aminophenyl 2-O-methyl-β-L-fucoside

78 mg (0.23 mmol) of p-nitrophenyl2-O-methyl-3,4-O-isopropylidene-β-L-fucoside are stirred in 3 ml of 80%strength acetic acid at 20° C. for 16 hours. The acetic acid is thenremoved in vacuo, 10 ml of methanol are added to the batch and, afteraddition of platinum dioxide, hydrogenation is carried out in a hydrogenatmosphere under a slightly increased pressure. The suspension isfiltered over Celite and the material on the filter is washed withmethanol. After purification by chromatography (methylenechloride/methanol 97.5:2.5), 77 mg (80%) of the target product areobtained. [TLC: methylene chloride/methanol 9:1 R_(f)=0.42].

EXAMPLE 1.2

p-Aminophenyl 3-O-methyl-β-L-fucoside

1.2.a) p-Nitrophenyl 3-O-Methyl-β-L-fucoside

784 g (31.5 mmol) of dibutyltin oxide are added to 6 g (21 mmol) ofp-nitrophenyl β-L-fucoside in 300 ml of absolute methanol and themixture is heated under reflux for 2 hours. It is then concentrated andthe residue is dried and then taken up in 300 ml of dimethylformamide.After addition of 15.7 ml of methyl iodide, the batch is stirred at 70°C. for 40 hours. The solvent is removed in vacuo and the residue istaken up in 300 ml of methylene chloride. The suspension is filtered,the solution which remains is concentrated again and the residue issubjected to flash chromatography (methylene chloride/methanol 99:1).After concentration, 381.5 mg (61%) of the target product are obtained.

1.2) p-Aminophenyl 3-O-methyl-β-L-fucoside

3.81 g (12.73 mol) of p-nitrophenyl 3-O-methyl-β-L-fucoside arehydrogenated analogously to example 1.1. Yield: 3 g (88%). [TLC:methylene chloride/methanol 9:1 R_(f)=0.53].

EXAMPLE 1.3

p-Aminophenyl 3-O-methyl-α-L-fucoside

Preparation analogous to Example 1.2 starting from p-nitrophenylα-L-fucoside. Yield: 63% over 2 stages. [TLC methylene chloride/methanol9:1 R_(f)=0.39].

EXAMPLE 1.4

p-Aminophenyl 4-O-methyl-β-L-fucoside

1.4.a) p-Nitrophenyl 2-O-benzyl-4-O-acetyl-β-L-fucoside

31 mg of p-toluenesulphonic acid and 1134 mg (7 mmol) of triethylorthoacetate are added to 1 g (3.5 mmol) of p-nitrophenyl β-L-fucosidein 100 ml of absolute tetrahydrofuran. After the mixture has beenstirred at 20° C. for 15 minutes, the solvent is distilled off in vacuo.The residue is taken up in 50 ml of tetrahydrofuran and 3 ml ofdimethylformamide, and 4165 μl of benzyl bromide and 210 mg of sodiumhydride (60% strength) are added. After the mixture has been stirred at20° C. for 1 hour, 10 ml of 80% strength acetic acid are added, themixture is concentrated and the residue is purified by flashchromatography (methylene chloride/methanol 99:1). After concentrationand drying, 1236 mg (85%) of the target product are obtained.

1.4.b) p-Nitrophenyl 2-O-benzyl-3-O-acetyl-4-O-methyl-β-L-fucoside

1000 mg (2.39 mmol) of p-nitrophenyl 2-O-benzyl-4-O-acetyl-β-L-fucosideare dissolved in 60 ml of benzene. After addition of 2988 βl of methyliodide and 1109 mg of silver oxide, the batch is heated under reflux for8 hours. The product mixture formed is separated into the components byflash chromatography (methylene chloride/methanol 99:1). 239 mg (23%) ofp-nitrophenyl 2-O-benzyl-3-O-acetyl-4-O-methyl-β-L-fucoside areisolated, in addition to 653 mg (63%) of the isomeric p-nitrophenyl2-O-benzyl-3-O-methyl-4-O-acetyl-β-L-fucoside, as a white solid.

1.4) p-Aminophenyl 4-O-methyl-β-L-fucoside

224 mg (0.52 mmol) of p-nitrophenyl2-O-benzyl-3-O-acetyl4-O-methyl-β-L-fucoside are dissolved in 20 ml ofmethanol, and 390 μl of a 1N sodium methylate solution are added. Afterthe mixture has been stirred at 20° C. for 16 hours, it is neutralizedwith 80% strength acetic acid and concentrated and the residue is takenup in methylene chloride. The organic phase is washed with 1N sodiumbicarbonate solution and with water, dried and concentrated. The residueis taken up in 20 ml of methanol and hydrogenated over palladium/activecharcoal analogously to Example 1.1. After concentration, the product istaken up in water and lyophilized. 119 mg (88%) of a white amorphoussolid are isolated. [TLC: methylene chloride/methanol 9:1 R_(f)=0.38].

EXAMPLE 1.5

p-Aminophenyl 3-O-n-propyl-β-L-fucoside

1.5.a) p-Nitrophenyl 2-O-benzyl-3-O-n-propyl-4-O-acetyl-β-L-fucoside

Analogously to Example 1.4.b, the isomeric 3- and 4-propylation productsare prepared from compound 1.4.a with propyl iodide and separated bychromatography. p-Nitrophenyl2-O-benzyl-3-O-n-propyl-4-O-acetyl-β-L-fucoside is obtained in a 49%yield, in addition to p-nitrophenyl2-O-benzyl-3-O-acetyl-4-O-n-propyl-β-L-fucoside in a 29% yield.

1.5.b) p-Aminophenyl 3-O-n-propyl-β-L-fucoside

Synthesis from Example 1.5.a) fraction 1 analogously to Example 1.4.Yield: 78% [TLC methylene chloride/methanol 9:1 R_(f)=0.42].

EXAMPLE 1.6

p-Aminophenyl 3-deoxy-β-L-fucoside

1.6.a) p-Nitrophenyl 3,6-dideoxy-3-chloro-4-O-acetyl-β-L-guloside

31 mg of p-toluenesulphonic acid and 1134 mg (7 mmol) of triethylorthoacetate are added to 1 g (3.5 mmol) of p-nitrophenyl-β-L-fucosidein 100 ml of tetrahydrofuran. After the mixture has been stirred at 20°C. for 15 minutes, the solvent is distilled off in vacuo. 100 ml of asaturated solution of hydrogen chloride in methylene chloride are added.After a reaction time of 10 minutes, the mixture is concentrated and theproduct is purified by flash chromatography (methylene chloride/methanol99:1). 793 mg (65%) of the target product are obtained. [TLC: methylenechloride/methanol 97.5:2.5 R_(f)=0.36].

1.6.b) p-Nitrophenyl 3,6-dideoxy-3-chloro-β-L-guloside

375 mg (1.08 mmol) of p-nitrophenyl3,6-dideoxy-3-chloro-4-O-acetyl-β-L-guloside are dissolved in 25 ml ofmethanol, and 10 drops of 1N sodium methylate solution are added. After20 minutes, the mixture is acidified with acetic acid and concentratedand the residue is partitioned between 400 ml of methylene chloride and60 ml of water. The organic phase is dried and concentrated and theresidue is precipitated from methylene chloride/ether. 315 mg (96%) ofthe target product are obtained.

1.6) p-Aminophenyl 3-deoxy-β-L-fucoside

315 mg (1.04 mmol) of p-nitrophenyl 3,6-dideoxy-3-chloro-β-L-gulosideare dissolved in 40 ml of methanol, 200 mg of palladium-on-activecharcoal and 290 μl of triethylamine are added and hydrogenation iscarried out in a hydrogen atmosphere under a slightly increased pressurefor 4 days. The suspension is filtered, washed and concentrated and theproduct is purified by flash chromatography (methylene chloride/methanol97.5:2.5). 160 mg (65%) of the deoxy compound are obtained. [TLC:methylene chloride/methanol 95:5 R_(f)=0.18].

EXAMPLE 1.7

p-Aminophenyl 3,4-dideoxy-β-L-fucoside

400 mg (1.16 mmol) of p-nitrophenyl3,6-dideoxy-3-chloro-4-O-acetyl-β-L-guloside (Example 1.6.a) aredissolved in 55 ml of methanol, 323 μl of triethylamine are added andhydrogenation is carried out in a hydrogen atmosphere under a slightlyincreased pressure over palladium/active charcoal (10%). After themixture has been stirred at 20° C. for 16 hours, it is filtered overCelite, the material on the filter is rinsed, the filtrate isconcentrated and the residue is taken again in 100 ml of methanol. 1.5ml of a 1N sodium methylate solution are added and the mixture isstirred at room temperature for 16 hours. It is neutralized with aceticacid and concentrated, and the products formed are separated by flashchromatography (methylene chloride/methanol 97.5:2.5). Afterconcentration of the corresponding fractions and reprecipitation frommethanol/ether, 120 mg (46%) of the target compound [TLC methylenechloride/methanol 95:5 R_(f)=0.31] are obtained, in addition to 77 mg(28%) of p-aminophenyl 3-deoxy-β-L-fucoside [TLC: methylenechloride/methanol 95:5 R_(f)=0.18].

EXAMPLE 1.8

p-Aminophenyl 3,4-epoxy-β-L-fucoside

80 mg (0.23 mmol) of p-nitrophenyl3,6-dideoxy-3-chloro-4-O-acetyl-β-L-guloside (Example 1.6.a) are takenup in 10 ml of methanol, and 345 μl of 1N sodium methylate solution areadded. After ultrasonic treatment for 1 hour, the mixture is acidifiedwith 80% strength acetic acid and concentrated and the residue ischromatographed with methylene chloride/methanol 99:1. Afterconcentration of the relevant fractions, the residue is taken up inmethanol and hydrogenation is carried out over palladium/active charcoalanalogously to Example 1.1. 46 mg (75%) of the target compound areobtained. FAB-MS: m/e=238=M+1.

EXAMPLE 1.9

p-Aminophenyl 4-deoxy-β-L-fucoside

This compound was prepared analogously to the instructions of T.Lindhorst and J. Thiem in Carbohydr. Res. 209 (1991), 119 starting fromp-nitrophenyl-β-L-fucoside via p-nitrophenyl2,3-di-O-benzoyl4,6-dideoxy4-iodo-β-L-fucoside. [TLC: methylenechloride/methanol 90:10 R_(f)=0.3].

EXAMPLE 1.10

p-Aminophenyl 3-O-carboxymethyl-β-L-fucoside

1.10.a) p-Nitrophenyl 3-O-methoxycarbonylmethyl-β-L-fucoside

1 g (3.5 mmol) of p-nitrophenyl β-L-fucoside and 1.3 g (5.2 mmol) ofdibutyltin oxide are heated under reflux in 50 ml of methanol for 2hours. The solution is concentrated, the residue is taken up in 50 ml ofdioxane, 2 ml of methyl bromoacetate and 100 mg of tetrabutylammoniumiodide are added and the mixture is heated under reflux for 16 hours.The solvent is evaporated off and the product is purified by flashchromatography (methylene chloride/methanol 99:1). After thecorresponding fractions have been concentrated and the residue has beenreprecipitated from methanol/ether, 455 mg (37%) of the target compoundare obtained.

1.10) p-Aminophenyl 3-O-carboxymethyl-β-L-fucoside

282 mg (0.79 mmol) of p-nitrophenyl 3-methoxycarbonylmethyl-β-L-fucosideare dissolved in 20 ml of methanol, and 440 μl of a 2N lithium hydroxidesolution are added. After the mixture has been stirred at 20° C. for 2hours, it is brought to pH 3 with acid ion exchanger SC108 and filtered.250 mg of palladium-on-active charcoal are added to the filtrate.Hydrogenation is then carried out with hydrogen under a slightlyincreased pressure for 1.5 hours and the catalyst is removed and washedwith methanol. Concentration of the mixture, taking up the residue inwater and freeze drying leads to the target product in an 86% yield (212mg). [TLC: acetonitrile/water/acetic acid 5:1:0.2 R_(f)=0.24].

EXAMPLE 1.11

p-Aminophenyl 3-O-methoxycarbonylmethyl-β-L-fucoside

250 mg (0.7 mmol) of p-nitrophenyl 3-methoxycarbonylmethyl-β-L-fucoside(Example 1.10.a) are dissolved in 20 ml of methanol and hydrogenation iscarried out with hydrogen over palladium-on-active charcoal under aslightly increased pressure for 1.5 hours. The catalyst is removed andwashed with methanol. Concentration, taking up in water and freezedrying lead to 195 mg (85%) of the target product. [TLC methylenechloride/methanol 9:1 R_(f)=0.43; FAB-MS: m/e=328=M+1.]

EXAMPLE 1.12

p-Aminophenyl 3-O-hydroxyethyl-β-L-fucoside

1.12.a) p-Nitrophenyl 3-O-hydroxyethyl-β-L-fucoside

1000 mg (2.8 mmol) of p-nitrophenyl 3-methoxycarbonylmethyl-β-L-fucosideare dissolved in a mixture of 160 ml of tetrahydrofuran and 40 ml ofwater, and 53 mg of sodium borohydride are added. After 10 minutes, thesolvent is evaporated off and the residue is purified by flashchromatography (methylene chloride/methanol 95:5). After thecorresponding fractions have been concentrated, the residue has beentaken up in water and the mixture has been freeze dried, 362 mg (40%) ofthe target product are obtained.

1.12) p-Aminophenyl 3-O-hydroxyethyl-β-L-fucoside

After hydrogenation of 362 mg of the compound from Example 1.12.a)analogously to Example 1.1, 270 mg (82%) of the target product areobtained. [TLC: acetonitrile/water 10:1 R_(f)=0.43].

EXAMPLE 1.13

p-Aminophenyl 2-O-carboxymethyl-β-L-fucoside

1.13.a) p-Nitrophenyl 2-O-methoxycarbonylmethyl-β-L-fucoside

250 mg (0.88 mmol) of p-nitrophenyl P-L-fucoside are dissolved in 25 mlof absolute tetrahydrofuran and 3 ml of dimethylformamide. 80 mg (2.64mmol) of 80% strength sodium hydride are added and, after the mixturehas been stirred at 20° C. for 10 minutes, 35 μl of benzyl bromoacetateare added. 3 further additions of 35 μl each of benzyl bromoacetate aremade at intervals of 10 minutes. The mixture is subsequently stirred for30 minutes and quenched with methanol. After a further 10 minutes, it isacidified with 5 ml of 80% strength acetic acid. The mixture isconcentrated and the residue is subsequently distilled with methylenechloride. Purification by means of flash chromatography is started withthe mobile phase system methylene chloride/methanol/glacial acetic acid90:10:1. The ratio in the same system is later changed to 80:20:2. Afterthe corresponding fractions have been concentrated, the residues aredigested with ether and 157 mg (42%) of the target compound are obtainedfrom the early eluate. [TLC: acetonitrile/water/glacial acetic acid5:1:0.2 R_(f)=0.65]. The isomeric 3-O-alkylated compound is obtainedfrom the late eluates (33%). [TLC: acetonitrile/water/glacial aceticacid 5:1:0.2 R_(f)=0.54].

1.13) p-Aminophenyl 2-O-carboxymethyl-β-L-fucoside

Hydrolysis and hydrogenation of 150 mg of p-nitrophenyl2-O-methoxycarbonylmethyl-β-L-fucoside by the procedure described inExample 1.10 leads to 109 mg of the target product in an 83% yield [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.35].

EXAMPLE 1.14.a

Synthesis of the regioisomeric monosuccinylation products ofp-nitrophenyl β-L-fucoside

1100 mg (3.86 mmol) of p-nitrophenyl β-L-fucoside are dissolved in 50 mlof pyridine, and 580 mg (5.79 mmol) of succinic anhydride are added.After the mixture has been stirred at 20° C. for 16 hours, it isconcentrated and the residue is subsequently distilled twice withmethylene chloride. The product is precipitated from methylenechloride/ether and 1 g of a substance mixture which cannot be separatedis obtained. This is taken up in methanol/water, and 846 mg (2.6 mmol)of caesium carbonate are added. The solvent is evaporated off and theresidue is subsequently distilled with dimethylformamide. The residue istaken up in dimethylformamide, and 618 μl of benzyl bromide are added.After ultrasonic treatment for 1 hour, the caesium bromide is filteredoff and the filtrate is concentrated. The residue is partitioned between500 ml of ethyl acetate and 50 ml of water. The organic phase is driedand concentrated. Separation of the components by flash chromatographyis achieved in the mobile phase system methylene chloride/methanol 99:1.This gives:

Fraction 1: 87 mg (4.8%) of p-nitrophenyl3-O-(3-benzyloxycarbonyl-propionyl)-β-L-fucoside [TLC: methylenechloride/methanol 95:5 R_(f)=0.45].

Fraction 2: 27 mg (1.5%) of p-nitrophenyl2-O-(3-benzyloxycarbonyl-propionyl)-β-L-fucoside [TLC: methylenechloride/methanol 95:5 R_(f)=0.34].

Fraction 3: 190 mg (10.3%) of p-nitrophenyl4-O-(3-benzyloxycarbonyl-propionyl)-β-L-fucoside [TLC: methylenechloride/methanol 95:5 R_(f)=0.28].

EXAMPLE 1.14

p-Aminophenyl 3-O-succinyl-β-L-fucoside

85 mg (0.17 mmol) of fraction 1 from Example 1.14.a are dissolved in 5ml of tetrahydrofuran and 1 ml of water. 20 mg of platinum dioxide areadded and hydrogenation is carried out for 8 hours. The catalyst isfiltered off and washed with tetrahydrofuran/water and the filtrate isconcentrated. The residue is taken up in water and lyophilized. 57 mg(94%) of the target product are obtained. [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.65].

EXAMPLE 1.15

p-Aminophenyl 2-O-succinyl-β-L-fucoside

Fraction 2 from Example 1.14.a is hydrogenated analogously to theinstructions in Example 1.14. Yield: 16 mg (87%) [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.62].

EXAMPLE 1.16

p-Aminophenyl 4-O-succinyl-β-L-fucoside

Fraction 3 from Example 1.14.a is hydrogenated analogously to theinstructions in Example 1.14. Yield: 125 mg (88%) [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.63].

EXAMPLE 1.17

p-Aminophenyl 3,4-di-O-methyl-β-L-fucoside

1.17.a) p-Nitrophenyl 2-O-benzyl-3,4-O-isopropylidene-β-L-fucoside

377 mg (1.16 mmol) of the compound from Example 1.1.a are dissolved in30 ml of absolute tetrahydrofuran, 690 μl of benzyl bromide and 52 mg ofsodium hydride are added in succession and the mixture is stirred at 20°C. The mixture is topped up with a further 690 μl of benzyl bromide andsodium hydride after 4 and 6 hours. The batch is worked up analogouslyto Example 1.1.b. 245 mg (51%) of the target compound are obtained.

1.17.b) p-Nitrophenyl 2-O-benzyl-3,4-di-O-methyl-β-L-fucoside

245 mg (0.59 mmol) of p-nitrophenyl2-O-benzyl-3,4-O-isopropylidene-β-L-fucoside are stirred in 80% strengthacetic acid at 20° C. for 16 hours. The mixture is concentrated and theresidue is stirred with ether/pentane. After the mixture have beenfiltered with suction and the residue dried, the product which remainsis taken up in 20 ml of absolute tetrahydrofuran, 45 mg of 80% strengthsodium hydride are added and, after 15 minutes, 160 μl of methyl iodideare injected in. After the mixture has been stirred at 20° C. for 20hours, it is quenched with methanol and glacial acetic acid andconcentrated and the residue is partitioned between methylene chlorideand water. The organic phase is dried and concentrated and the residueis then purified by flash chromatography (methylene chloride/methanol100:1). After concentration and drying of the corresponding fractions,188 mg (79%) of the target product are obtained.

1.17) p-Aminophenyl 3,4-di-O-methyl-β-L-fucoside

180 mg (0.45 mmol) of the compound from Example 1.17.b are hydrogenatedin a mixture of 15 ml of methanol and 3 ml of methylene chloride at roomtemperature for 2 days, after addition of 50 mg of palladium-on-activecharcoal. The catalyst is filtered off, the filtrate is concentrated andthe residue is purified by flash chromatography (methylenechloride/methanol 97.5:2.5). 86 mg (68%) of the target compound areobtained. [TLC: methylene chloride/methanol 95:5 R_(f)=0.21].

EXAMPLE 1.18

p-Aminophenyl 3-O-carbamoylmethyl-β-L-fucoside

100 mg (0.305 mmol) of the compound from Example 1.11 are dissolved in10 ml of methanol, and 0.5 ml of 17% strength ammonium hydroxidesolution is added. After 4 hours, the mixture is concentrated and theresidue is taken up in water and lyophilized. 95 mg (quantitative) ofthe target compound are obtained. [TLC: acetonitrile/water 10:1R_(f)=0.43].

EXAMPLE 1.19

p-Aminophenyl 2-O-hydroxyethyl-β-L-fucoside

This compound was prepared analogously to Examples 1.12.a and 1.12starting from 200 mg (0.56 mmol) of p-nitrophenyl2-O-methoxycarbonylmethyl-β-L-fucoside (Example 1.13.a). Yield: 76 mg(45% over 2 stages) [TLC: methylene chloride/methanol 9:1 R_(f)=0.2].

EXAMPLE 1.20

p-Aminophenyl 3,6-dideoxy-3-chloro-β-L-guloside

50 mg (0.165 mmol) of the compound from Example 1.6.b are hydrogenatedin 5 ml of methanol over palladium/active charcoal for 1 hour. Thecatalyst is filtered off and rinsed, the filtrate is concentrated andthe residue is taken up in water and lyophilized. 45 mg (89%) of thetarget compound are obtained. [TLC: methylene chloride/methanol 9:1R_(f)=0.35].

EXAMPLE 1.21

p-Aminophenyl α-L-rhamnoside

This compound was prepared analogously to Example 1.1 starting from 300mg of p-nitrophenyl α-L-rhamnoside (Sigma). Yield: 96%

EXAMPLE 1.22

p-Aminophenyl 3-O-carboxymethyl-α-L-rhamnoside

1.22.a) p-Nitrophenyl 3-O-methoxycarbonylmethyl-α-L-rhamnoside

481 mg (1.63 mmol) of p-nitrophenyl α-L-rhamnoside are taken up in 30 mlof methanol, and 629 mg (2.45 mmol) of dibutyltin oxide are added. Themixture is heated under reflux for 2 hours and concentrated and theresidue is taken up in 30 ml of dioxane. 85 mg of tetrabutyl-ammoniumiodide and 950 μl of methyl bromoacetate are added and the mixture isheated under reflux for 16 hours. If appropriate, it is topped up with afurther 1 ml of methyl bromoacetate and the reaction time is prolonged.The mixture is concentrated and the residue is purified by flashchromatography. p-Nitrophenyl 3-O-methoxycarbonylmethyl-α-L-rhamnosideis eluted with methylene chloride/methanol 99:1 and, after drying, 408mg (70%) are obtained. [TLC: methylene chloride/methanol 95:5R_(f)=0.36].

1.22) p-Aminophenyl 3-O-carboxymethyl-α-L-rhamnoside

Synthesis completely analogously to Example 1.10 starting fromp-nitrophenyl 3-O-methoxycarbonylmethyl-α-L-rhamnoside. The targetproduct is obtained in an 80% yield. [TLC: acetonitrile/water/glacialacetic acid 5:1:0.2 R_(f)=0.26].

EXAMPLE 1.23

p-Aminophenyl β-D-galactopyranoside

p-Nitrophenyl β-D-galactopyranoside (3.0 g, 10 mmol) is dissolved inmethanol/water 1:1 (50 ml) and, after addition of palladium-on-activecharcoal (10% of Pd, 200 mg), hydrogenation is carried out in a hydrogenatmosphere under a slightly increased pressure for 3 hours. Thesuspension is filtered over Celite and the material on the filter iswashed with hot methanol/water 1:1 (100 ml). Concentration of thefiltrate in vacuo and recrystallization from methanol gives colourlesscrystals (2.11 g, 78%); TLC [methanol]: R_(f)=0.62; [α]²⁰=−39.5°(c=1.0/H₂O); melting point=166° C.

EXAMPLE 1.24

p-Aminophenyl 2-O-methyl-β-D-galactopyranoside

1.24.a) p-Nitrophenyl 6-O-triphenylmethyl-β-D-galactopyranoside

A solution of p-nitrophenyl β-D-galactopyranoside (9.0 g, 30 mmol),chlorotriphenylmethane (16.7 g, 60 mmol) and N,N-dimethylaminopyridine(609 mg, 5 mmol) in absolute pyridine (100 ml) is heated at 60° C. for 4hours. After concentration in vacuo, the residue is purified by flashchromatography [petroleum ether/ethyl acetate 2:1→3:2, in each case with0.5% of triethylamine]. Colourless crystals (9.23 g, 57%) are obtained;TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.55;melting point=82° C.

1.24.b) p-Nitrophenyl3,4-O-isopropylidene-6-O-triphenylmethyl-β-D-galactopyranoside

Dimethoxypropane (400 ml) and a catalytic amount of(±)-camphor-10-sulphonic acid (400 mg, 1.7 mmol) are added to the abovecompound (8.7 g, 16 mmol). After 1 hour at room temperature, thereaction is ended by addition of triethylamine (240 ml, 1.7 mmol) andthe mixture is concentrated in vacuo. Flash chromatography [petroleumether/ethyl acetate 2:1] gives a colourless foam (6.2 g, 66%); TLC[petroleum ether/ethyl acetate 1:1]: R_(f)=0.46; [α]²⁰=−42.1°(c=0.94/CH₂Cl₂).

1.24.c) p-Nitrophenyl2-O-methyl-3,4-O-isopropylidene-6-O-triphenylmethyl-β-D-galactopyranoside

Compound 1.24.b (5.83 g, 10 mmol) is dissolved in dimethylformamide (100ml), and methyl iodide (2.5 ml, 40 mmol) and, in portions, an 80%strength suspension of sodium hydride in mineral oil (450 mg, 15 mmol)are added. After 2 hours at room temperature, the reaction is ended bydropwise addition of methanol (10 ml) and the mixture is concentrated invacuo. The residue is taken up in methylene chloride (1000 ml) and thesolution is stirred vigorously with water (500 ml). The organic phase isdried over magnesium sulphate (50 g) and concentrated in vacuo and theresidue is purified by flash chromatography [petroleum ether/ethylacetate 12:1→8:1]. A colourless foam (4.72 g, 79%) is obtained; TLC[petroleum ether/ethyl acetate 1:1]: R_(f)=0.72; [α]²⁰=−35.7°(c=1.0/CH₃OH).

1.24.d) p-Nitrophenyl 2-O-methyl-β-D-galactopyranoside

99% strength trifluoroacetic acid (20 ml) is added to a solution of theabove compound (4.48 g, 7.5 mmol) in methylene chloride (200 ml) and themixture is stirred at room temperature for 3 hours. After the mixturehas been concentrated in vacuo, the residue is purified by flashchromatography [petroleum ether/ethyl acetate 5:1→2:1]. Colourlesscrystals (1.09 g, 46%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.42; melting point177° C.

1.24) p-Aminophenyl 2-O-methyl-β-D-galactopyranoside

Compound 1.24.d (946 mg, 3 mmol) is dissolved in methanol (50 ml) and,after addition of water (0.5 ml) and basic Raney nickel (about 200 mg),hydrogenation is carried out in a hydrogen atmosphere under a slightlyincreased pressure for 2 hours. The suspension is filtered over Celiteand the material on the filter is washed thoroughly with methanol (100ml). Concentration of the filtrate in vacuo gives a brownish foam (579mg, 68%); TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.28; [α]²⁰=−39.3° (c=0.15/CH₃OH).

EXAMPLE 1.25

p-Aminophenyl 3-O-methyl-β-D-galactopyranoside

1.25.a) p-Nitrophenyl 3-O-methyl-β-D-galactopyranoside

Dibutyltin oxide (1.87 g, 7.5 mmol) is added to a solution ofp-nitrophenyl β-D-galactopyranoside (1.5 g, 5.0 mmol) in absolutemethanol (40 ml) and the mixture is heated under reflux. After 3 hours,it is concentrated in vacuo and the residue is dried under an oil pumpvacuum for 1 hour. It is taken up in absolute dioxane (40 ml), methyliodide (1.9 ml, 30 mmol) is added to the resulting solution and thebatch is stirred at a bath temperature of 100° C. for 16 hours. Thesolvent is then distilled off in vacuo and the residue is purified byflash chromatography [ethyl acetate/petroleum ether 2:1→ethyl acetate].Colourless crystals (1.32 g, 84%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.34; meltingpoint=196° C.; [α]²⁰=−53.3° (c=1.0/CH₃OH).

1.25) p-Aminophenyl 3-O-methyl-β-D-galactopyranoside

The above compound (946 mg, 3 mmol) is reduced as described in Example1.24 and the product is worked up. Brownish crystals (656 mg, 77%) areobtained; TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.21; melting point=196° C.; [α]²⁰=−25.2° (c=1.0/CH₃OH).

EXAMPLE 1.26

p-Aminophenyl 4-O-methyl-β-D-galactopyranoside, acetate

1.26.a) p-Nitrophenyl 3-O-benzyl-β-D-galactopyranoside

Dibutyltin oxide (1.87 g, 7.5 mmol) is added to a solution ofp-nitrophenyl β-D-galactopyranoside (1.5 g, 5.0 mmol) in absolutedioxane (40 ml) and the mixture is heated under reflux. After 3 hours,benzyl bromide (3.6 ml, 30 mmol) are added to the solution obtained andthe batch is stirred under reflux for a further 48 hours. The solvent isthen distilled off in vacuo and the residue is purified by flashchromatography (ethyl acetate/petroleum ether 2:1→1:1]. Colourlesscrystals (1.58 g 81%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.69; meltingpoint=127° C.

1.26.b) p-Nitrophenyl3-O-benzyl-4,6-O-isopropylidene-β-D-galactopyranoside

Compound 1.26.a (6.26 g, 16 mmol) is reacted as described in Example1.24.b. After flash chromatography [petroleum ether/ethyl acetate5:1→3:1], a colourless foam (6.54 g, 95%) is obtained; TLC [petroleumether/ethyl acetate 1:1]: R_(f)=0.34; [α]²⁰=−38.9° (c=1.0/CH₂Cl₂).

1.26.c) p-Nitrophenyl2,3-di-O-benzyl-4,6-O-isopropylidene-β-D-galactopyranoside

Compound 1.26.b (4.31 g, 10 mmol) is dissolved in dimethylformamide (100ml), and benzyl bromide (12 ml, 100 mmol) and, in portions, an 80%strength suspension of sodium hydride in mineral oil (450 mg, 15 mmol)are added. After 2 hours at room temperature, the reaction is ended bydropwise addition of methanol (10 ml) and the mixture is concentrated invacuo. The residue is taken up in methylene chloride (1000 ml) and thesolution is stirred vigorously with water (500 ml). The organic phase isdried over magnesium sulphate (50 g) and concentrated in vacuo and theresidue is purified by flash chromatography [petroleum ether/ethylacetate 20:1→15:1→10:1]. A colourless oil (2.72 g, 52%), which is stillcontaminated, is obtained; TLC [petroleum ether/ethyl acetate 1:1]:R_(f)=0.62.

1.26.d) p-Nitrophenyl 2,3-di-O-benzyl-β-D-galactopyranoside

The above compound (2.6 g, 5 mmol) is reacted as described in Example1.24.d. After concentration in vacuo and extraction of the residue byboiling with diethyl ether, colourless crystals (805 mg, 33%) areobtained; TLC [petroleum ether/ethyl acetate 1:1]: R_(f)=0.23; meltingpoint=160° C.

1.26.e) p-Nitrophenyl2,3-di-O-benzyl-6-O-triphenylmethyl-β-D-galactopyranoside

Compound 1.26.d (722 mg, 1.5 mmol) is tritylated as described in Example1.24.a. After flash chromatography [petroleum ether/ethyl acetate15:1→10:1→5:1], a colourless foam (880 mg, 81%) is obtained; TLC[petroleum ether/ethyl acetate 2:1]: R_(f)=0.79; [α]²⁰=−25.3°(c=0.3/CH₂Cl₂).

1.26.f) p-Nitrophenyl2,3-di-O-benzyl-4-O-methyl-6-O-triphenylmethyl-β-D-galactopyranoside

The above compound (724 mg, 1 mmol) is methylated as described inExample 1.24.c. After flash chromatography [petroleum ether/ethylacetate 10:1→5:1], a colourless foam (662 mg, 90%) is obtained; TLC[petroleum ether/ethyl acetate 5:1]: R_(f)=0.66; [α]²⁰=−38.7°(c=0.2/CH₂Cl₂).

1.26) p-Aminophenyl 4-O-methyl-β-D-galactopyranoside, acetate

The above compound (590 mg, 0.8 mmol) is dissolved in 90% strengthacetic acid (50 ml) and, after addition of palladium-on-active charcoal(10% of Pd, 200 mg), hydrogenation is carried out in a hydrogenatmosphere under a slightly increased pressure for 16 hours. Thesuspension is filtered over Celite and the material on the filter iswashed thoroughly with methanol (100 ml). Concentration of the filtratein vacuo and reprecipitation of the residue from diethyl ether/petroleumether gives colourless crystals (253 mg, 92%); TLC [methylenechloride/methanol 5:1]: R_(f)=0.12.

EXAMPLE 1.27

p-Aminophenyl 6-O-methyl-β-D-galactopyranoside

1.27.a) Benzylation of compound 1.24.b

Compound 1.24.b (5.84 g, 10 mmol) is benzylated as described in Example1.26.c. After flash chromatography [petroleum ether/ethyl acetate15:1→12:1→5:1→ethyl acetate, in each case with 0.5% of triethylamine],two product fractions are obtained:

Fraction 1: p-nitrophenyl2-O-benzyl-3,4-O-isopropylidene-6-O-triphenylmethyl-β-D-galactopyranoside;yellowish foam (1.71 g, 25%); TLC [petroleum ether/ethyl acetate 2:1]:R_(f)=0.72; [α]²⁰=−8.1° (c=1.0/CH₂Cl₂).

Fraction 2: p-nitrophenyl2-O-benzyl-3,4-O-isopropylidene-β-D-galactopyranoside; yellowish oil(806 g, 19%); TLC [petroleum ether/ethyl acetate 2:1]: R_(f)=0.45;[α]²⁰=+2.8° (c=1.2/CH₃OH).

1.27.b) p-Nitrophenyl2-O-benzyl-3,4-O-isopropylidene-6-O-methyl-β-D-galactopyranoside

Fraction 2 from Example 1.27.a (777 mg, 1.8 mmol) is methylated asdescribed in Example 1.24.c. After flash chromatography [petroleumether/ethyl acetate 10:1→8:1], a brownish oil (730 mg, 91%) is obtained;TLC [petroleum ether/ethyl acetate 2:1]: R_(f)=0.54; [α]²⁰=−11.6°(c=1.1/CH₂Cl₂).

1.27.c) p-Nitrophenyl 2-O-benzyl-6-O-methyl-β-D-galactopyranoside

The above compound (668 mg, 1.5 mmol) is reacted as described in Example1.24.d. Concentration of the filtrate in vacuo and extraction of theresidue by boiling with a little diethyl ether gives, after cooling toroom temperature, pale beige crystals (388 mg, 64%); TLC [petroleumether/ethyl acetate 1:1]: R_(f)=0.15; melting point=143° C.

1.27) p-Aminophenyl 6-O-methyl-β-D-galactopyranoside

Compound 1.27.c (324 mg, 0.8 mmol) is reduced for 16 hours as describedin Example 1.24. After concentration of the filtrate in vacuo andextraction of the residue by boiling with a little diethyl ether, beigecrystals (184 mg, 81%) are obtained; TLC [ethyl acetate]: R_(f)=0.05;melting point=115° C. (decomposition).

EXAMPLE 1.28

p-Aminophenyl 2,3-di-O-methyl-β-D-galactopyranoside

1.28.a) Isopropylidenation of p-nitrophenyl β-D-galactopyranoside

Anhydrous toluenesulphonic acid (500 mg) is added to a solution ofp-nitrophenyl β-D-galactopyranoside (7.5 g, 25 mmol) in absolute acetone(1000 ml). Acetone (250 ml) is distilled off under normal pressure inthe course of 30 minutes and, immediately thereafter, the mixture isneutralized by addition of potassium carbonate (500 mg). Afterconcentration in vacuo, the residue is stirred with diethyl ether (1000ml). The mixture is filtered, the filtrate is concentrated and theresidue is purified by flash chromatography [petroleum ether/ethylacetate 2:1→1:1→3:2]. Two product fractions are obtained by thisprocedure:

Fraction 1: p-Nitrophenyl 3,4-O-isopropylidene-β-D-galactopyranoside;colourless foam (3.74 g, 44%); TLC [methylene chloride/methanol/ammonia(25%) 15:3:0.2]: R_(f)=0.59; [α]²⁰=−54.2° (c=0.38/CH₃OH).

Fraction 2: p-Nitrophenyl 4,6-O-isopropylidene-β-D-galactopyranoside;colourless foam (4.3 g, 50%); TLC [methylene chloride/methanol/ammonia(25%) 15:3:0.2]: R_(f)=0.54; [α]²⁰=−81.0° (c=0.31/CH₃OH).

1.28.b) p-Nitrophenyl2,3-di-O-methyl-4,6-O-isopropylidene-β-D-galactopyranoside

Fraction 2 from Example 1.28.a (4.1 g, 12 mmol) is methylated asdescribed in Example 1.24.c. After flash chromatography [petroleumether/ethyl acetate 5:1→2:1], a colourless foam (2.93 g, 66%) isobtained; TLC [[petroleum ether/ethyl acetate 1:1]: R_(f)=0.42;[α]²⁰=−52.6° (c=0.34/CH₃OH).

1.28.c) p-Nitrophenyl 2,3-di-O-methyl-β-D-galactopyranoside

The above compound (2.77 g, 7.5 mmol) is reacted as described in Example1.24.d. After 1 hour at room temperature, the mixture is concentrated invacuo and the residue is dried under an oil pump vacuum for 1 hour.Digestion with diethyl ether/petroleum ether 1:1 (100 ml) givescolourless crystals (1.11 g, 45%); TLC [ethyl acetate]: R_(f)=0.24;melting point=156° C.

1.28) p-Aminophenyl 2,3-di-O-methyl-β-D-galactopyranoside

Compound 1.28.c (989 mg, 3 mmol) is reduced as described in Example1.24. A colourless oil (396 mg, 44%) is obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.50; [α]²⁰=−19.4°(c=0.16/CH₃OH).

EXAMPLE 1.29

p-Aminophenyl 2,4-di-O-methyl-β-D-galactopyranoside

1.29.a) Tritylation of Compound 1.26.a

Compound 1.26.a (11.7 g, 30 mmol) is tritylated as described in Example1.24.a. After flash chromatography [petroleum ether/ethyl acetate10:1→7:1→5.1, in each case with 0.5% of triethylamine], two products areobtained:

Fraction 1: triphenylmethyl2-O-(p-nitrophenyl)-3-O-benzyl-6-O-triphenylmethyl-β-D-galactopyranoside,colourless foam (8.5 g, 32%); TLC [petroleum ether/ethyl acetate 2:1]:R_(f)=0.68; [α]²⁰=+42.8° (c=1.0/CH₂Cl₂).

Fraction 2: p-nitrophenyl3-O-benzyl-6-O-triphenylmethyl-β-D-galactopyranoside, colourless foam(9.0 g, 47%); TLC [petroleum ether/ethyl acetate 2:1]: R_(f)=0.22;[α]²⁰=−22.6° (c=1.03/CH₂Cl₂).

1.29.b) p-Nitrophenyl2,4-di-O-methyl-3-O-benzyl-6-O-triphenylmethyl-β-D-galactopyranoside

Fraction 2 from Example 1.29.a (7.6 g, 12 mmol) is methylated asdescribed in Example 1.24.c. After flash chromatography [petroleumether/ethyl acetate 15:1→10:1], a colourless foam (7.07 g, 89%) isobtained; TLC [petroleum ether/ethyl acetate 2:1]: R_(f)=0.79;[α]²⁰=−35.8° (c=1.09/CH₃OH).

1.29.c) p-Aminophenyl 2,4-di-O-methyl-3-O-benzyl-β-D-galactopyranoside

The above compound (6.0 g, 9 mmol) is hydrogenated for 48 hours asdescribed in Example 1.26. Colourless crystals (1.39 g, 40%) areobtained; TLC [ethyl acetate/petroleum ether 2:1]: R_(f)=0.20; meltingpoint=148° C.

1.29) p-Aminophenyl 2,4-di-O-methyl-β-D-galactopyranoside

Compound 1.29.c (779 mg, 2 mmol) is hydrogenated for 5 days as describedin

Example 1.24. After evaporation of the combined filtrates in vacuo andextraction of the residue by boiling with diethyl ether (2×50 ml),slightly greenish crystals (391 mg, 65%) are obtained; TLC [ethylacetate]: R_(f)=0.16; melting point=260° C. (decomposition).

EXAMPLE 1.30

p-Aminophenyl 2,6-di-O-methyl-β-D-galactopyranoside

1.30.a) p-Nitrophenyl2,6-di-O-methyl-3,4-O-isopropylidene-β-D-galactopyranoside

Fraction 1 from Example 1.28.a (4.1 g, 12 mmol) is methylated asdescribed in Example 1.24.c. After flash chromatography [petroleumether/ethyl acetate 10:1→8:1→5:1], a colourless oil (3.25 g, 73%) isobtained; TLC [petroleum ether/ethyl acetate 1:1]: R_(f)=0.65.

1.30.b) p-Nitrophenyl 2,6-di-O-methyl-β-D-galactopyranoside

The above compound (2.77 g, 7.5 mmol) is reacted as described in Example1.24.d. After flash chromatography [ethyl acetate/petroleum ether 2:1],colourless crystals (1.63 g, 66%) are obtained; TLC [ethyl acetate]:R_(f)=0.31; melting point=222° C.

1.30) p-Aminophenyl 2,6-di-O-methyl-β-D-galactopyranoside

Compound 1.30.b (989 mg, 3 mmol) is reduced as described in Example1.24. A colourless oil (597 mg, 66%) is obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.56; [α]²⁰=−53.1°(c=0.49/CH₃OH).

EXAMPLE 1.31

p-Aminophenyl 3,4-di-O-methyl-β-D-galactopyranoside, acetate

1.31.a) p-Nitrophenyl2,6-di-O-benzyl-3,4-O-isopropylidene-β-D-galactopyranoside

Fraction 1 from Example 1.28.a (4.1 g, 12 mmol) is benzylated asdescribed in Example 1.26.c. After flash chromatography [petroleumether/ethyl acetate 6:1], a yellowish oil (5.3 g, 85%) is obtained; TLC[ethyl acetate/petroleum ether 2:1]: R_(f)=0.76; [α]²⁰=+8.8°(c=1.2/CH₃OH).

1.31.b) p-Nitrophenyl 2,6-di-O-benzyl-β-D-galactopyranoside

The above compound (4.69 g, 9 mmol) is reacted as described in Example1.24.d. After 30 minutes at room temperature, the mixture isconcentrated in vacuo and the residue is dried under an oil pump vacuumfor 1 hour. Recrystallization from ethanol gives colourless crystals(2.89 g, 67%); TLC [ethyl acetate/petroleum ether 2:1]: R_(f)=0.42;melting point=133° C.; [α]²⁰=−64.2° (c=1.0/CH₃OH).

1.31.c) p-Nitrophenyl2,6-di-O-benzyl-3,4-di-O-methyl-β-D-galactopyranoside

The above compound (2.4 g, 5 mmol) is methylated as described in Example1.24.c. After recrystallization from ethanol/n-hexane, colourlesscrystals (1.74 g, 69%) are obtained; TLC [petroleum ether/ethyl acetate1:1]: R_(f)=0.74; melting point=149° C.

1.31) p-Aminophenyl 3,4-di-O-methyl-β-D-galactopyranoside, acetate

Compound 1.31.c (1.52 g, 3 mmol) is hydrogenated as described in Example1.26. Colourless crystals (664 mg, 62%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.47; meltingpoint=140° C. (decomposition).

EXAMPLE 1.32

p-Aminophenyl 3,6-di-O-methyl-β-D-galactopyranoside

1.32.a) p-Nitrophenyl3-O-methyl-6-O-(tert-butyldimethylsilyl)-β-D-galactopyranoside

Imidazole (1 g, 15 mmol) and tert-butyldimethylsilyl chloride (1.25 g, 8mmol) are added to a solution of compound 1.25.a (1.58 g, 5 mmol) indimethylformamide (150 ml) and the mixture is stirred at roomtemperature for 24 hours. The reaction is then interrupted by additionof water (100 ml). The mixture is diluted with methylene chloride (1000ml) and the organic phase is washed with water (2×1000 ml), dried overmagnesium sulphate (20 g) and concentrated in vacuo. After flashchromatography [petroleum ether/ethyl acetate 15:1→10:1→5:11, ayellowish foam (826 mg, 38%), which is still slightly contaminated, isobtained; TLC [ethyl acetate]: R_(f)=0.59; [α]²⁰=−56.3° (c=1.0/CH₂Cl₂).

1.32.b) p-Nitrophenyl2,4-di-O-benzyl-3-O-methyl-6-O-(tert-butyldimethylsilyl)-β-D-galactopyranoside

The above compound (773 mg, 1.8 mmol) is benzylated as described inExample 1.26.c. After flash chromatography [petroleum ether/ethylacetate 30:1→5:1], a colourless foam (810 mg, 74%) is obtained; TLC[petroleum ether/ethyl acetate 5:1]: R_(f)=0.58.

1.32.c) p-Nitrophenyl 2,4-di-O-benzyl-3-O-methyl-β-D-galactopyranoside

Compound 1.32.b (732 mg, 1.2 mmol) is dissolved in tetrahydrofuran (6ml) and a 1M solution of tetrabutylammonium fluoride in tetrahydrofuran(2.4 ml) is added at 0° C. The mixture is stirred at room temperaturefor 40 minutes and then concentrated in vacuo. After flashchromatography [petroleum ether/ethyl acetate 5:1→3:1→2:1], colourlesscrystals (512 mg, 86%) are obtained; TLC [petroleum ether/ethyl acetate1:1]: R_(f)=0.36; melting point=177° C.

1.32.d) p-Nitrophenyl2,4-di-O-benzyl-3,6-di-O-methyl-β-D-galactopyranoside

The above compound (446 mg, 0.9 mmol) is methylated as described inExample 1.24.c. After flash chromatography [petroleum ether/ethylacetate 20:1→10:1→8:1, a colourless oil (401 mg, 87%) is obtained; TLC[petroleum ether/ethyl acetate 1:1]: R_(f)=0.70; [α]²⁰=−56.5°(c=0.96/CH₂Cl₂).

1.32) p-Aminophenyl 3,6-di-O-methyl-β-D-galactopyranoside

Compound 1.32.d (357 mg, 0.7 mmol) is hydrogenated as described inExample 1.24. After evaporation of the combined filtrates in vacuo andextraction of the residue by boiling with diethyl ether (20 ml),colourless crystals (207 mg, 99%) are obtained; TLC [petroleumether/ethyl acetate 1:1]: R_(f)=0.02; melting point=>280° C.(decomposition).

EXAMPLE 1.33

p-Aminophenyl 4,6-di-O-methyl-βD-galactopyranoside

1.33.a) p-Nitrophenyl2,3-di-O-benzyl-4,6-di-O-methyl-β-D-galactopyranoside

Compound 1.26.d (2.4 g, 5 mmol) is methylated as described in Example1.24.c. After flash chromatography [petroleum ether/ethyl acetate5:1→3:1], colourless crystals (1.89 g, (74%) are obtained; TLC[petroleum ether/ethyl acetate 1:1]: R_(f)=0.76; melting point=177° C.

1.33) p-Aminophenyl 4,6-di-O-methyl-β-D-galactopyranoside

Compound 1.33.a (1.53 g, 3 mmol) is hydrogenated as described in Example1.24. Colourless crystals (890 mg, 99%) are obtained; TLC[methanol/ethyl acetate 1:1]: R_(f)=0.71; melting point=180° C.(decomposition).

EXAMPLE 1.34

p-Aminophenyl 2,3,4-tri-O-methyl-β-D-galactopyranoside

1.34.a) p-Nitrophenyl2,3,4-tri-O-methyl-6-O-triphenylmethyl-β-D-galactopyranoside

Compound 1.24.a (1.63 g, 3 mmol) is methylated as described in Example1.24.c. After flash chromatography [petroleum ether/ethyl acetate5:1→3:1], a colourless foam (1.24 g, 71%) is obtained; TLC [petroleumether/ethyl acetate 1:1]: R_(f)=0.54; [α]²⁰=−53.6° (c=0.3/CH₃OH).

1.34.b) p-Nitrophenyl 2,3,4-tri-O-methyl-β-D-galactopyranoside

The above compound (1.17 g, 2 mmol) is reacted as described in Example1.24.d. After flash chromatography [petroleum ether/ethyl acetate3:1→2:1], colourless crystals (468 mg, 68%) are obtained; TLC [petroleumether/ethyl acetate 1:1]: R_(f)=0.12; melting point=104° C.;[α]²⁰=−68.2° (c=0.47/CH₃OH).

1.34) p-Aminophenyl 2,3,4-tri-O-methyl-β-D-galactopyranoside

Compound 1.34.b (343 mg, 1 mmol) is reduced as described in Example1.24. Beige crystals (224 mg, 71%) are obtained; TLC (methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.67; meltingpoint=138° C.

EXAMPLE 1.35

p-Aminophenyl 2,3,6-tri-O-methyl-β-D-galactopyranoside

1.35.a) p-Nitrophenyl 2,3,6-tri-O-methyl-β-D-galactopyranoside

Compound 1.30.b (2.63 g, 3 mmol) is methylated selectively as describedin Example 1.25.a. After flash chromatography [petroleum ether/ethylacetate 5:1→2:1], a brownish oil (890 mg, 32%) is obtained; TLC [ethylacetate]: R_(f)=0.37; [α]²⁰=−63.3° (c=0.9/CH₂Cl₂).

1.35) p-Aminophenyl 2,3,6-tri-O-methyl-β-D-galactopyranoside

The above compound (858 mg, 2.5 mmol) is reduced as described in Example1.24. A beige foam (519 mg, 66%) is obtained; TLC [ethyl acetate]:R_(f)0.23; [α]²⁰=−34.5° (c=0.86/CH₃OH).

EXAMPLE 1.36

p-Aminophenyl 2,4,6-O-methyl-β-D-galactopyranoside

1.36.a) p-Nitrophenyl2,4,6-tri-O-methyl-3-O-benzyl-β-D-galactopyranoside

Compound 1.26.a (1.96 g, 5 mmol) is methylated as described in Example1.24.c. After flash chromatography [petroleum ether/ethyl acetate10:1→8:1], colourless crystals (1.47 g, 68%) are obtained; TLC[petroleum ether/ethyl acetate 2:1]: R_(f)=0.46; melting point=164° C.

1.36) p-Aminophenyl 2,4,6-tri-O-methyl-β-D-galactopyranoside

The above compound (1.3 g, 3 mmol) is reduced as described in Example1.26. Colourless crystals (642 mg, 68%) are obtained; TLC [ethylacetate/petroleum ether 2:1]: R_(f)=0.12; melting point=147° C.(decomposition).

EXAMPLE 1.37

p-Aminophenyl 3,4,6-tri-O-methyl-β-D-galactopyranoside

1.37.a) p-Nitrophenyl 2-O-benzyl-β-D-galactopyranoside

Fraction 1 from Example 1.27.a (1.17 g, 2 mmol) is reacted as describedin Example 1.24.d. After flash chromatography [petroleum ether/ethylacetate 3:1→2:1], colourless crystals (468 mg, 68%) are obtained; TLC[petroleum ether/ethyl acetate 1:1]: R_(f)=0.12; melting point=104° C.;[α]²⁰=−68.2° (c=0.47/CH₃OH).

1.37.b) p-Nitrophenyl2-O-benzyl-3,4,6-tri-O-methyl-β-D-galactopyranoside

The above compound (391 mg, 1 mmol) is methylated as described inExample 1.24.c. After flash chromatography [petroleum ether/ethylacetate 10:1→5:1], pale yellow crystals (303 mg, 70%) are obtained; TLC[ethyl acetate]: R_(f)=0.81; [α]²⁰=−76.5° (c=1.1/CH₂Cl₂).

1.37) p-Aminophenyl 3,4,6-tri-O-methyl-β-D-galactopyranoside

Compound 1.37.b (260 mg, 0.6 mmol) is hydrogenated as described inExample 1.24. Beige crystals (161 mg, 86%) are obtained; TLC [ethylacetate]: R_(f)=0.20; melting point=132° C.

EXAMPLE 1.38

p-Aminophenyl 2,3,4,6-tetra-O-methyl-β-D-galactopyranoside

1.38.a) p-Nitrophenyl 2,3,4,6-tetra-O-methyl-β-D-galactopyranoside

p-Nitrophenyl β-D-galactopyranoside (904 mg, 3 mmol) is methylated asdescribed in Example 1.24.c. After flash chromatography [petroleumether/ethyl acetate 8:1→6:1→4:1→2:1], a colourless, waxy solid (633 mg,59%) is obtained; TLC [ethyl acetate]: R_(f)=0.67; [α]²⁰=−55.7°(c=0.9/CH₂Cl₂).

1.38) p-Aminophenyl 2,3,4,6-tetra-O-methyl-β-D-galactopyranoside

Compound 1.33.a (536 mg, 1.5 mmol) is hydrogenated as described inExample 1.24. After evaporation of the combined filtrates in vacuo andextraction of the residue by boiling with diethyl ether (20 ml),colourless crystals (412 mg, 84%) are obtained; TLC [ethyl acetate]:R_(f)=0.42; melting point=204° C. (decomposition).

EXAMPLE 1.39

p-Aminophenyl α-D-mannopyranoside

p-Nitrophenyl α-D-mannopyranoside (3.0 g, 10 mmol) is hydrogenated asdescribed in Example 1.23. The precipitation from methanol/diethyl ethergives colourless crystals (2.03 g, 75%); TLC [methanol]: R_(f)=0.69;[α]²⁰=+102.7° (c=1.0/H₂O); melting point=161° C.

EXAMPLE 1.40

p-Aminophenyl 3-O-methyl-α-D-mannopyranoside

1.40.a) p-Nitrophenyl 6-O-triphenylmethyl-α-D-mannopyranoside

p-Nitrophenyl α-D-mannopyranoside (3.0 g, 10 mmol) is tritylated asdescribed in Example 1.24.a. Colourless crystals (4.35 g, 80%) areobtained; TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.52; [α]²⁰=+104.0° (c=1.0/CH₃OH); melting point=102-104° C.

1.40.b) p-Nitrophenyl 3-O-methyl-6-O-triphenylmethyl-α-D-mannopyranoside

The above compound (2.72 g, 5 mmol) is reacted with methyl iodide (2 ml,30 mmol) as described in Example 1.26.a. After flash chromatography[petroleum ether/ethyl acetate 2:1] and reprecipitation fromethanol/n-hexane, colourless crystals (1.83 g, 66%) are obtained; TLC[methylene chloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.68;[α]²⁰=+106.4° (c=1.0/CH₃OH); melting point=104° C.

1.40) p-Aminophenyl 3-O-methyl-α-D-mannopyranoside

Compound 1.40.b (1.4 g, 2.5 mmol) is dissolved in methanol (50 ml) and,after addition of palladium-on-active charcoal (10% of Pd, 300 mg),hydrogenation is carried out in a hydrogen atmosphere under a slightlyincreased pressure for 24 hours. The suspension is filtered over Celiteand the material on the filter is washed thoroughly with methanol (100ml). After concentration of the filtrate in vacuo, the residue isextracted with water (50 ml), the mixture is filtered and the filtrateis lyophilized. A brownish amorphous solid (709 mg, 99%) is obtained;TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.33;[α]²⁰=+92.9° (c=1.1/CH₃OH).

EXAMPLE 1.41

p-Aminophenyl 2,3-di-O-methyl-α-D-mannopyranoside

1.41.a) p-Nitrophenyl 4,6-O-benzylidene-α-D-mannopyranoside

Benzaldehyde dimethyl acetal (3.2 ml, 21.4 mmol) and a 54% strengthsolution of tetrafluoboric acid in diethyl ether (2.7 ml, 20 mmol) areadded to a solution of p-nitrophenyl α-D-mannopyranoside (6.0 g, 20mmol) in dimethylformamide (120 ml). The mixture is stirred at roomtemperature for 5 hours, the reaction is then interrupted by addition oftriethylamine (2.8 ml, 20 mmol) and the mixture is concentrated invacuo. After flash chromatography [toluene→toluene/ethanol 20:1],colourless crystals (6.48 g, 83%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.82; [α]²⁰=+170.7°(c=1.0/CH₂Cl₂); melting point=116° C.

1.41.b) p-Nitrophenyl2,3-di-O-methyl-4,6-O-benzylidene-α-D-mannopyranoside

The above compound (3.9 g, 10 mmol) is methylated as described inExample 1.24.c. After flash chromatography [petroleum ether/ethylacetate 20:1→7:1] and reprecipitation from ethyl acetate/n-hexane, acolourless foam (3.2 g, 77%) is obtained; TLC [ethyl acetate/petroleumether 2:1]: R_(f)=0.67; [α]²⁰=+167.30 (c=1.05/CH₃OH).

1.41) p-Aminophenyl 2,3-di-O-methyl-α-D-mannopyranoside

Compound 1.41.b (1.25 g, 3 mmol) is hydrogenated as described in Example1.26. After flash chromatography [ethyl acetate/petroleum ether2:1→ethyl acetate, in each case with 0.5% of triethylamine], areddish-brown foam (480 mg, 53%) is obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.31; [α]²⁰=+83.6°(c=0.76/CH₃OH).

EXAMPLE 1.42

1.42.a) p-Nitrophenyl 3-O-methoxycarbonylmethyl-α-D-galactopyranoside

Dibutyltin oxide (9.3 g, 37.5 mmol) is added to a solution ofp-nitrophenyl β-D-galactopyranoside (7.53 g, 25 mmol) in absolutedioxane (180 ml) and the mixture is heated under reflux. After 4 hours,methyl bromoacetate (8.3 ml, 90 mmol) and tetrabutylammonium iodide(9.25 g, 25 mmol) are added to the solution obtained and the batch isstirred under reflux for a further 3 hours. The solvent is thendistilled off in vacuo and the residue is purified by flashchromatography [methylene chloride/methanol 50:1→20:1]. In addition tosome by-products, the compound 1.42.a is obtained as colourless crystals(4.05 g, 43%); TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.54; [α]²⁰=−62.0° (c=1.0/CH₃OH); melting point 176° C.

1.42) p-Aminophenyl 3-O-methoxycarbonylmethyl-β-D-galactopyranoside

Compound 1.42.a (3.73 g, 10 mmol) is hydrogenated as described inExample 1.24. After reprecipitation from ethanol/-hexane, colourlesscrystals (2.98 g, 87%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.39; [α]²⁰=−36.3°(c=1.07/CH₃OH); melting point=155° C.

EXAMPLE 1.43

1.43.a) p-Nitrophenyl 3-O-carboxymethyl-β-D-galactopyranoside, sodiumsalt

A solution of sodium hydroxide (400 mg, 10 mmol) in water (5 ml) isadded to a solution of compound 1.42.a (3.73 g, 10 mmol) in methanol(100 ml) and the mixture is stirred at room temperature for 2 hours.After concentration in vacuo, the residue is dried under an oil pumpvacuum for 2 hours and ethanol (100 ml) is then added. The mixture isboiled under reflux for 5 minutes and, after cooling in an ice-bath, thesolid is filtered off to give colourless crystals (3.66 g, 96%); TLC[methanol]: R_(f)=0.62; [α]²⁰=−50.0° (c=1.0/CH₃OH); meltingpoint=180-185° C.

1.43) p-Aminophenyl 3-O-carboxymethyl-β-D-galactopyranoside, sodium salt

The above compound (3.05 g, 8 mmol) is hydrogenated as described inExample 1.24. After extraction by boiling with ethanol (50 ml),colourless crystals (2.03 g, 72%) are obtained; TLC [methanol]:R_(f)=0.70; [α]²⁰=−22.4° (c=1.0/CH₃OH); melting point=180-182° C.

EXAMPLE 1.44

p-Aminophenyl 3-O-carbamoylmethyl-β-D-galactopyranoside

1.44.a) p-Nitrophenyl 3-O-carbamoylmethyl-β-D-galactopyranoside

A 25% strength aqueous solution of ammonia (10 ml) is added to asolution of compound 1.42.a (373 mg, 1 mmol) in methanol (30 ml) and themixture is stirred at room temperature for 15 minutes. Afterconcentration in vacuo, the residue is dried under an oil pump vacuumfor 2 hours and ethanol (30 ml) is then added. The mixture is boiledunder reflux for 5 minutes and filtered, after cooling in an ice-bath,to give colourless crystals (306 mg, 85%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.14; [α]²⁰=−41.7°(c=1.0/CH₃OH); melting point=229° C. (decomposition).

1.44) p-Aminophenyl 3-O-carbamoylmethyl-β-D-galactopyranoside

The above compound (287 mg, 0.8 mmol) is hydrogenated as described inExample 1.24. After reprecipitation from methanol/diethyl ether,colourless crystals (207 mg, 79%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.10; meltingpoint=205° C. (decomposition).

EXAMPLE 1.45

p-Aminophenyl 3-O-(N-methyl-carbamoylmethyl)-β-D-galactopyranoside

1.45a) p-Nitrophenyl3-O-(N-methyl-carbamoylmethyl)-β-D-galactopyranoside

A 30% strength aqueous solution of methylamine (10 ml) is added to asolution of compound 1.42.a (373 mg, 1 mmol) in methanol (30 ml) and themixture is stirred at room temperature for 2 hours. After concentrationin vacuo, the residue is dried under an oil pump vacuum for 2 hours andthen recrystallized from ethanol. Colourless crystals (372 mg, 100%) areobtained; TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.33; [α]²⁰=−36.7° (c=1.0/CH₃OH); melting point=205° C.

1.45) p-Aminophenyl 3-O-(N-methyl-carbamoylmethyl)-β-D-galactopyranoside

Compound 1.45.a (298 mg, 0.8 mmol) is hydrogenated as described inExample 1.24. After reprecipitation from methanol/diethyl ether,colourless crystals (180 mg, 66%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.16; meltingpoint=239° C.

EXAMPLE 1.46

p-Aminophenyl 3-O-(N-propyl-carbamoylmethyl)-β-D-galactopyranoside

1.46.a) p-Nitrophenyl3-O-(N-propyl-carbamoylmethyl)-β-D-galactopyranoside

Compound 1.42.a (373 mg, 1 mmol) is reacted with n-propylamine (823 μl,10 mmol) as described in Example 1.45.a. After concentration in vacuo,the residue is reprecipitated from ethanol/n-hexane. Colourless crystals(340 mg, 85%) are obtained; TLC [methylene chloride/methanol/ammonia(25%) 15:3:0.2]: R_(f)=0.49; [α]²⁰=−32.4° (c=1.0/CH₃OH); meltingpoint=155° C.

1.46) p-Aminophenyl ³-O-(N-propyl-carbamoylmethyl)-β-D-galactopyranoside

Compound 1.46.a (320 mg, 0.8 mmol) is hydrogenated as described inExample 1.24. After reprecipitation from methanol/diethyl ether,colourless crystals (188 mg, 63%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.31; meltingpoint=154° C.

EXAMPLE 1.47

p-Aminophenyl 3-O-(N-butyl-carbamoylmethyl)-β-D-galactopyranoside

1.47.a) p-Nitrophenyl3-O-(N-butyl-carbamoylmethyl)-β-D-galactopyranoside

Compound 1.42.a (373 mg, 1 mmol) is reacted with n-butylamine (900 μl,10 mmol) as described in Example 1.45.a. After concentration in vacuo,the residue is reprecipitated from ethanol/n-hexane. Colourless crystals(413 mg, 100%) are obtained; TLC [methylene chloride/methanol/ammonia(25%) 15:3:0.2]: R_(f)=0.51; [α]²⁰=−26.8° (c=1.0/CH₃OH); meltingpoint=92° C.

1.47) p-Aminophenyl 3-O-(N-butyl-carbamoylmethyl)-β-D-galactopyranoside

Compound 1.47.a (332 mg, 0.8 mmol) is hydrogenated as described inExample 1.24. After reprecipitation from ethanol/n-hexane, colourlesscrystals (105 mg, 34%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.32; meltingpoint=135° C.

EXAMPLE 1.48

p-Aminophenyl 3-O-methoxycarbonylmethyl-α-D-mannopyranoside

1.48.a) p-Nitrophenyl3-O-methoxycarbonylmethyl-6-O-triphenylmethyl-α-D-mannopyranoside

Compound 1.40.a (13.6 g, 25 mmol) is reacted as described in Example1.42.a. After flash chromatography [petroleum ether/ethyl acetate 10:1],in addition to some by-products, colourless crystals (2.79 g, 18%) areobtained; TLC [ethyl acetate/petroleum ether 2:1]: R_(f)=0.50; meltingpoint=95-97° C.

1.48) p-Aminophenyl 3-O-methoxycarbonylmethyl-α-D-mannopyranoside

Compound 1.48.a (1.23 g, 2 mmol) is hydrogenated as described in Example1.40 and the product is worked up. A brownish amorphous solid (250 mg,36%) is obtained; TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.45.

EXAMPLE 1.49

p-Aminophenyl 3-O-carboxymethyl-α-D-mannopyranoside

1.49.a) p-Nitrophenyl3-O-benzoxycarbonylmethyl-6-O-triphenylmethyl-α-D-mannopyranoside

Compound 1.40.a (13.6 g, 25 mmol) is reacted with benzyl bromoacetate(14.4 ml, 90 mmol) as described in Example 1.42.a. After flashchromatography [petroleum ether/ethyl acetate 20:1→10:1], in addition tosome by-products, a yellowish foam (5.0 g, 29%) is obtained; TLC [ethylacetate/petroleum ether 2:1]: R_(f)=0.66; [α]²⁰=+74.8° (c=1.0/CH₂Cl₂).

1.49) p-Aminophenyl 3-O-carboxymethyl-α-D-mannopyranoside

The above compound (2.08 g, 3 mmol) is hydrogenated for 36 hours asdescribed in Example 1.40. After concentration of the filtrate, theresidue is reprecipitated from ethanol/n-hexane. Washing with ethylacetate and renewed reprecipitation from ethanol/diethyl ether givescolourless crystals (495 mg, 50%); TLC [methanol]: R_(f)=0.53; meltingpoint=205-207° C.

EXAMPLE 1.50

p-Aminophenyl 3-O-carbamoylmethyl-α-D-mannopyranoside

1.50.a) p-Nitrophenyl3-O-carbamoylmethyl-6-O-triphenylmethyl-α-D-mannopyranoside

Compound 1.49.a (1.04 g, 1.5 mmol) is reacted as described in Example1.44.a. After drying under an oil pump vacuum, the residue is purifiedby flash chromatography [petroleum ether/ethyl acetate 2:3]. Colourlesscrystals (561 mg, 62%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.67; [α]²⁰=+91.3°(c=1.0/CH₂Cl₂); melting point=125-127° C.

1.50) p-Aminophenyl 3-O-carbamoylmethyl-α-D-mannopyranoside

The above compound (541 g, 0.9 mmol) is hydrogenated for 48 hours asdescribed in Example 1.40. After concentration of the filtrate, theresidue is washed thoroughly with methanol to give colourless crystals(134 mg, 45%); TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.21; melting point=126-128° C.

EXAMPLE 1.51

p-Aminophenyl 3-deoxy-β-D-galactopyranoside

1.51.a) p-Nitrophenyl 2,6-di-O-benzyl-4-O-acetyl-β-D-galactopyranoside

Triethyl orthoacetate (3 ml, 16.3 mmol) and toluenesulphonic acid (20mg) are added to a solution of compound 1.31.b (2.7 g, 5.6 mmol) inmethylene chloride (20 ml). After 30 minutes at room temperature, thebatch is diluted with methylene chloride (200 ml) and washed withsaturated sodium bicarbonate solution (50 ml), the organic phase isdried over magnesium sulphate and, after filtration, the filtrate isconcentrated in vacuo. The resulting colourless foam is dissolved in 80%strength acetic acid (15 ml). After a further 30 minutes at roomtemperature, the batch is poured into saturated sodium bicarbonatesolution (200 ml) and extracted with chloroform (3×75 ml), the combinedorganic phases are washed with water (100 ml) and dried over magnesiumsulphate and, after filtration, the filtrate is concentrated in vacuo.Reprecipitation from ethanol/petroleum ether gives colourless crystals(2.43 g, 83%); TLC [ethyl acetate/petroleum ether 2:1]: R_(f)=0.64;[α]²⁰=−62.1° (c=1.0/CH₂Cl₂); melting point=83° C.

1.51.b) p-Nitrophenyl2,6-di-O-benzyl-3-O-trifluoromethanesulphonyl-4-O-acetyl-β-D-galactopyranoside

A solution of trifluoromethanesulphonic anhydride (2 ml, 11.8 mmol) inmethylene chloride (30 ml) is added dropwise to a solution of compound1.51.a (2.3 g, 4.4 mmol) in a mixture of methylene chloride (30 ml) andpyridine (3 ml) at −20° C., under argon. After 1 hour at −20° C., thebatch is poured into saturated sodium bicarbonate solution (200 ml), theorganic phase is separated off and dried over magnesium sulphate and,after filtration, the filtrate is concentrated in vacuo. After flashchromatography [toluene→toluene/ethyl acetate 20:1], colourless crystals(2.39 g, 83%) are obtained; TLC [toluene/ethyl acetate 5:1]: R_(f)=0.55;[α]²⁰=−61.4° (c=1.0/CH₂Cl₂); melting point=105° C.

1.51.c) p-Nitrophenyl 2,6-di-O-benzyl-3-deoxy-β-D-galactopyranoside

The above compound (1.31 g, 2 mmol) is dissolved in toluene (25 ml), andtetrabutylammonium tetraborohydrate (1.54 g, 6 mmol) is added. After 2hours at 80° C., the batch is diluted with methylene chloride (200 ml)and washed once with water (50 ml), the organic phase is dried overmagnesium sulphate and, after filtration, the filtrate is concentratedin vacuo. After flash chromatography [toluene/ethyl acetate 7:1],colourless crystals (596 mg, 64%) are obtained; TLC [toluene/ethylacetate 5:1]: R_(f)=0.10; [α]²⁰=−81.3° (c=1.0/CH₂Cl₂); meltingpoint=114° C.

1.51) p-Aminophenyl 3-deoxy-β-D-galactopyranoside

Compound 1.51.c (465 mg, 1 mmol) is hydrogenated for 6 hours asdescribed in Example 1.40. After concentration of the filtrate, theresidue is reprecipitated from ethanol/petroleum ether to givecolourless crystals (206 mg, 81%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.22.

EXAMPLE 1.52

p-Aminophenyl 3,4-dideoxy-β-D-galactopyranoside

1.52.a) p-Nitrophenyl2,6-di-O-benzyl-3,4-di-O-trifluoromethanesulphonyl-β-D-galactopyranoside

Compound 1.31.b (2.12 g, 4.4 mmol) is reacted withtrifluoromethanesulphonic anhydride (4 ml, 23.6 mmol) as in Example1.51.b. After flash chromatography [toluene→toluene/ethyl acetate 50:1],a yellowish oil (2.75 g, 84%) is obtained; TLC [toluene/ethyl acetate5:1]: R_(f)=0.67; [α]²⁰=−22.5° (c=1.0/CH₂Cl₂).

1.52.b) p-Nitrophenyl 2,6-di-O-benzyl-3,4-dideoxy-β-D-galactopyranoside

Compound 1.52.a (1.49 g, 2 mmol) is reacted with tetrabutylammoniumtetraborohydrate (2.31 g, 9 mmol) as in Example 1.51.c. After flashchromatography [toluene→toluene/ethyl acetate 50:1], colourless crystals(629 mg, 70%) are obtained; TLC [toluene/ethyl acetate 5:1]: R_(f)=0.53;[α]²⁰=−79.1° (c=1.0/CH₂Cl₂); melting point=89° C.

1.52) p-Aminophenyl 3,4-dideoxy-β-D-galactopyranoside

Compound 1.52.b (450 mg, 1 mmol) is hydrogenated for 5 hours asdescribed in Example 1.40. After concentration of the filtrate, theresidue is reprecipitated from ethanol/petroleum ether to givecolourless crystals (183 mg, 76%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.47; [α]²⁰=115.1°(c=1.0/CH₃OH); melting point=187° C.

EXAMPLE 1.53

p-Aminophenyl 6-O-acetyl-β-D-galactopyranoside

1.53.a) p-Nitrophenyl 6-O-acetyl-β-D-galactopyranoside

A freshly prepared solution of pyridine (2 ml, 25 mmol) and acetylchloride (1.85 ml, 26 mmol) in acetonitrile (20 ml) is added dropwise toa solution of p-nitrophenyl β-D-galactopyranoside (7.53 g, 25 mmol) inabsolute acetonitrile (80 ml) at 0° C. The mixture is stirred at 0° C.for 30 minutes and then concentrated in vacuo.

After flash chromatography [methylene chloride/methanol 50:1→20:1],colourless crystals (4.02 g, 47%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.50.

1.53) p-Aminophenyl 6-O-acetyl-β-D-galactopyranoside

Compound 1.53.a (1.72 g, 5 mmol) is hydrogenated for 2 hours asdescribed in Example 1.40. After concentration of the filtrate, theresidue is reprecipitated from methanol/diethyl ether to give colourlesscrystals (1.34 g, 86%); TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.34; [α]²⁰=41.0° (c=0.56/CH₃OH); melting point=180° C.(decomposition).

EXAMPLE 1.54

p-Aminophenyl 3,4-di-O-methoxycarbonylmethyl-β-D-galactopyranoside

1.54.a) Acylation of compound 1.24.a

Compound 1.24.a (1.36 g, 3 mmol) is dissolved in dimethylformamide (25ml) and methyl bromoacetate (1 ml, 10.6 mmol) and, in portions, an 80%strength suspension of sodium hydride in mineral oil (300 mg, 10 mmol)are added. After 3.5 hours at room temperature, methyl bromoacetate (250μl, 2.65 mmol) and sodium hydride in mineral oil (75 mg, 2.5 mmol) areagain added. After a further 2 hours, the reaction is ended by dropwiseaddition of methanol (5 ml) and the mixture is concentrated in vacuo.The residue is taken up in methylene chloride (250 ml) and the solutionis stirred vigorously with water (100 ml). The organic phase is driedover magnesium sulphate (10 g) and concentrated in vacuo and the residueis purified by flash chromatography [petroleum ether/ethyl acetate10:1→7:1→5:1→3:1]. Three product fractions are obtained:

Fraction 1: p-nitrophenyl2,3,4-tri-O-methoxycarbonylmethyl-β-D-galactopyranoside; colourless foam(401 mg, 21%); TLC [petroleum ether/ethyl acetate 1:1]: R_(f)=0.47;[α]²⁰=−51.9° (c=0.26/CH₃OH).

Fraction 2: not identified; colourless foam (88 mg); TLC [petroleumether/ethyl acetate 1:1]: R_(f)=0.39; [α]²⁰=−61.5° (c=0.26/CH₃OH).

Fraction 3: p-nitrophenyl3,4-di-O-methoxycarbonylmethyl-β-D-galactopyranoside; colourless foam(275 mg, 16%); TLC [petroleum ether/ethyl acetate 1:1]: R_(f)=0.30;[α]²⁰=−38.6° (c=0.28/CH₃OH).

1.54) p-Aminophenyl 3,4-di-O-methoxycarbonylmethyl-β-D-galactopyranoside

Fraction 3 from Example 1.54.a (206 mg, 0.3 mmol) is hydrogenated for 16hours as described in Example 1.40. After concentration of the filtrate,the residue is extracted by boiling with diethyl ether (20 ml) to givegrey crystals (45.6 mg, 37%); TLC [petroleum ether/ethyl acetate 1:1]:R_(f)=0.22; melting point=155° C. (decomposition).

EXAMPLE 1.55

p-Aminophenyl 2,3,4-tri-O-methoxycarbonylmethyl-β-D-galactopyranoside

Fraction 1 from Example 1.54.a (380 mg, 0,5 mmol) is hydrogenated for 16hours as described in Example 1.40. After concentration of the filtrate,the residue is extracted by boiling with diethyl ether (20 ml) to give ayellow-brown oil (46.8 mg, 19%); TLC [petroleum ether/ethyl acetate1:1]: R_(f)=0.29; melting point=106° C. (decomposition).

EXAMPLE 1.56

p-Aminophenyl 4-O-(β-D-galactopyranosyl)-β-D-glucopyranoside

p-Nitrophenyl 4-O-(β-D-galactopyranosyl)-β-D-galactopyranoside (4.63 g,10 mmol) is hydrogenated as described in Example 1.23. Colourlesscrystals (3.04 g, 70%) are obtained; TLC [methanol]: R_(f)=0.55, meltingpoint=235-237° C. (decomposition).

EXAMPLE 1.57

p-Aminophenyl4-O-(3′-sulphato-β-D-galactopyranosyl)-β-D-glucopyranoside, sodium salt

1.57.a) p-Nitrophenyl4-O-(3′,4′-O-isopropylidene-β-D-galactopyranosyl)-β-D-glucopyranoside

Dimethoxypropane (400 ml) and a catalytic amount of(±)-camphor-10-sulphonic acid (400 mg, 1.7 mmol) are added top-nitrophenyl 4-O-(β-D-galactopyranosyl)-β-D-galactopyranoside (23.2 g,50 mmol). After 3 days at room temperature, the reaction is ended byaddition of triethylamine (240 μl, 1.7 mmol), the mixture isconcentrated in vacuo and the residue is dried under an oil pump vacuumfor 2 hours. The resulting crystals are taken up in methanol/water 10:1(500 ml) and the mixture is boiled under reflux for 6 hours. Afterconcentration in vacuo and flash chromatography [methylenechloride/methanol 25:1→10:1, in each case with 0.5% of triethylamine],colourless crystals (15.2 g, 60%) are obtained; TLC [methylenechloride/methanol 5:1]: R_(f)=0.49; melting point=253-255° C.(decomposition).

1.57.b) p-Nitrophenyl2,3,6-tri-O-benzoyl-4-O-(2′,6′-di-O-benzoyl-3′,4′-O-isopropylidene-β-D-galactopyranosyl)-β-D-glucopyranoside

Benzoyl chloride (50 ml, 430 mmol) is slowly added dropwise to asolution of compound 1.57.a (15.1 g, 30 mmol) in pyridine (300 ml) at 0°C. in the course of 30 minutes. The mixture is then stirred at roomtemperature for a further 2 hours and the batch is subsequently pouredinto ice-water (2000 ml), while stirring. After 15 minutes, the crystalswhich have precipitated out are filtered off and taken up in methylenechloride (1500 ml). The solution is washed with water (2×500 ml) and 1Nsodium bicarbonate solution (2×500 ml), the organic phase is dried overmagnesium sulphate (50 g) and concentrated in vacuo and the residue ispurified by recrystallization from methanol. Colourless crystals (26.7g, 87%) are obtained; TLC [methylene chloride/methanol 50:1]:R_(f)=0.49; [α]²⁰=+23.6° (c=1.08/CH₂Cl₂); melting point=272-274° C.

1.57.c) p-Nitrophenyl2,3,6-tri-O-benzoyl-4-O-(2′,6′-di-O-benzoyl-β-D-galactopyranosyl)-β-D-glucopyranoside

99% strength trifluoroacetic acid (20 ml) is added to a solution ofcompound 1.57.b (20.5 g, 20 mmol) in methylene chloride (400 ml) and themixture is stirred at room temperature for 20 minutes. The solution isthen washed with 1N sodium bicarbonate solution (2×200 ml), the organicphase is dried over magnesium sulphate (10 g) and concentrated in vacuoand the residue is purified by reprecipitation from methylenechloride/diethyl ether. Colourless crystals (18.0 g, 91%) are obtained;TLC [methylene chloride/methanol 20:1]: R_(f)=0.18; melting point=234°C.

1.57.d) p-Nitrophenyl2,3,6-tri--benzoyl-4-O-(2′,6′-di-O-benzoyl-4′-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside

Compound 1.57.c (5.5 g, 5.6 mmol) is reacted as described in Example1.51.a. After flash chromatography [petroleum ether/ethyl acetate3:1→1:1], colourless crystals (4.03 g, 70%) are obtained; TLC [methylenechloride/methanol 20:1]: R_(f)=0.67; melting point=118° C.

1.57.e) p-Nitrophenyl2,3,6-tri-O-benzoyl4-O-(2′,6′-di-O-benzoyl-3′-sulphato-4′-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside,sodium salt

Sulphur trioxide-pyridine complex (4.5 g, 28 mmol) is added to asolution of compound 1.57.d (3.59 g, 3.5 mmol) in pyridine (200 ml) andthe mixture is stirred first at 60° C. for 2 hours and then at roomtemperature for 16 hours. The reaction is then ended by dropwiseaddition of methanol (50 ml) and the mixture is concentrated in vacuo.The residue is purified by flash chromatography [methylenechloride/methanol 10:1]. A solid product is obtained and is taken up inmethylene chloride/methanol 1:1 (200 ml), and Amberlite IR120 (Na⁺ form,10 g) is added. This mixture is stirred at room temperature for 1 hourand filtered and the filtrate is concentrated in vacuo. Colourlesscrystals (3.64 g, 92%) are obtained; TLC [methylene chloride/methanol2:1]: R_(f)=0.87; melting point=168° C.

1.57.f) p-Nitrophenyl4-O-(3′-sulphato-β-D-galactopyranosyl)-β-D-glucopyranoside, sodium salt

Compound 1.57.e (3.4 g, 3 mmol) is dissolved in absolute methanol (150ml), sodium methylate (200 mg) is added and the mixture is stirred at60° C. for 7 hours. After cooling to room temperature, the mixture isneutralized with Lewatit SC108 (H⁺ form) and then filtered. The pH ofthe filtrate is increased to pH 7-8 by dropwise addition of IN sodiumhydroxide solution, the mixture is evaporated in vacuo andreprecipitation of the residue from methanol/diethyl ether givesslightly brownish crystals (1.30 g, 77%); TLC [methylenechloride/methanol 2:1]: R_(f)=0.55; melting point=230° C.(decomposition).

1.57) p-Aminophenyl4-O-(3′-sulphato-β-D-galactopyranosyl)-β-D-glucopyranoside, sodium salt

The above compound (1.13 g, 2 mmol) is reduced as described in Example1.24. After extraction of the residue by boiling with diethyl ether (50ml), colourless crystals (983 mg, 92%) are obtained; TLC [methylenechloride/methanol 2:1]: R_(f)=0.22; melting point=176° C.(decomposition).

EXAMPLE 1.58

p-Aminophenyl 4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside

1.58.a) Selective methylation of p-nitrophenyl4-O-(β-D-galactopyranosyl)-β-D-glucopyranoside

p-Nitrophenyl 4-O-(β-D-galactopyranosyl)-β-D-glucopyranoside (2.3 g, 5mmol) is methylated as described in Example 1.25.a. Flash chromatography[methylene chloride/methanol 20:1→10:1→5:1] gives two products:

Fraction 1: p-nitrophenyl2-O-methyl-4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside;colourless crystals (264 mg, 11%); TLC [methylene chloride/methanol5:1]: R_(f)=0.46; [α]²⁰=−73.9° (c=1.0/CH₃OH); melting point=228° C.(decomposition).

Fraction 2: p-nitrophenyl4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside; colourlesscrystals (1.0 g, 42%); TLC [methylene chloride/methanol 5:1]:R_(f)=0.29; [α]²⁰=−65.3° (c=1.1/CH₃OH); melting point=220° C.

1.58) p-Aminophenyl4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside

Fraction 2 from Example 1.58.a (955 mg, 2 mmol) is reduced as describedin Example 1.24. After washing with diethyl ether (50 ml), colourlesscrystals (894 mg, 100%) are obtained; TLC [methylene chloride/methanol5:1]: R_(f)=0.08; melting point=129° C. (decomposition).

EXAMPLE 1.59

p-Aminophenyl2-O-methyl-4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside

Fraction 1 from Example 1.58.a (246 mg, 0.5 mmol) is reduced asdescribed in Example 1.24. After washing with diethyl ether (20 ml),colourless crystals (186 mg, 81%) are obtained; TLC [methylenechloride/methanol 5:1]: R_(f)=0.13; [α]²⁰=−3.6° (c=1.0/CH₃OH); meltingpoint=105° C.

EXAMPLE 1.60

p-Aminophenyl4-O-(3′,4′-di-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside

1.60.a) p-Nitrophenyl2,3,6-tri-O-benzyl-4-O-(2′,6′-di-O-benzyl-3′,4′-O-isopropylidene-β-D-galactopyranosyl)-β-D-glucopyranoside

Compound 1.57.a (5.0 g, 10 mmol) is reacted with benzyl bromide (30 ml,250 mmol) for 16 hours as described in Example 1.26.c. Afterconcentration in vacuo, the residue is taken up in ethyl acetate (300ml) and the solution is washed with water (200 ml). The organic phase isdried over magnesium sulphate (10 g) and concentrated in vacuo and theresidue is purified by flash chromatography [methylenechloride/petroleum ether 5:1→methylene chloride]. A brownish oil (5.3 g,56%) is obtained; TLC [methylene chloride/methanol 50:1]: R_(f)=0.70;[α]²⁰=−23.2° (c=1.08/CH₂Cl₂).

1.60.b) p-Nitrophenyl2,3,6-tri-O-benzyl-4-O-(2′,6′-di-O-benzyl-5-D-galactopyranosyl)-β-D-glucopyranoside

The above compound (4.77 g, 5 mmol) is reacted as described in Example1.57.c. After concentration in vacuo and reprecipitation from diethylether/petroleum ether, colourless crystals (3.94 g, 86%) are obtained;TLC [methylene chloride/methanol 50:1]: R_(f)=0.36; melting point=116°C.

1.60.c) p-Nitrophenyl2,3,6-tri-O-benzyl-4-O-(2′,6′-di-O-benzyl-3′,4′-di-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside

Compound 1.60.b (1.8 g, 2 mmol) is methylated as described in Example1.24.c. Reprecipitation from methylene chloride/petroleum ether givescolourless crystals (1.55 g, 82%); TLC [methylene chloride/methanol50:1]: R_(f)=0.74; melting point=161-162° C.

1.60) p-Aminophenyl4-O-(3′,4′-di-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranoside

Compound 1.60.c (1.41 g, 1.5 mmol) is dissolved in methanol (50 ml) andafter addition of palladium hydroxide-on-charcoal (moist, 20% of Pd, 500mg), hydrogenation is carried out in a hydrogen atmosphere under aslightly increased for 6 days. The suspension is filtered over Celiteand the material on the filter is washed thoroughly with methanol (100ml). Concentration of the filtrate in vacuo and washing of the residuewith methylene chloride gives brownish crystals (425 mg, 61%); meltingpoint=124° C. (decomposition).

EXAMPLE 2.1

N-Alanyl-batracyline

2.1.a) N-[N-(tert-Butoxycarbonyl)-alanyl]-batracyline

N-(tert-Butoxycarbonyl)-alanine (3.3 g, 17.5 mmol) and2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (6.8 ml, 23 mmol)are dissolved in 100 ml of methylene chloride. After the mixture hasbeen stirred at room temperature for 20 minutes, a solution ofbatracyline (4.1 g, 16.5 mmol) in absolute dimethylformamide (350 ml) isadded and the batch is stirred at room temperature for a further 48hours. It is then concentrated to 50 ml in vacuo and the concentrate istopped up to 300 ml with ethyl acetate and immediately heated at theboiling point for 10 minutes. The mixture is then allowed to cool toroom temperature and is filtered and the material on the filter isextracted by boiling again with ethyl acetate (200 ml). Cooling to 0°C., while stirring, and filtration gives yellow crystals (6.18 g, 84%);TLC [ethyl acetate]: R_(f)=0.57; melting point=246-247° C.(decomposition).

2.1) N-Alanyl-batracyline

A solution of compound 2.1.a (10.5 g, 25 mmol) in anhydroustrifluoroacetic acid (150 ml) is stirred at room temperature for 15minutes. After the batch has been concentrated to 30 ml in vacuo, it ispoured into saturated sodium bicarbonate solution (1000 ml) whilestirring vigorously. Stirring is continued for 10 minutes, the mixtureis filtered and the residue is washed with water, a little isopropanoland diethyl ether. The product is obtained in yellow crystals (7.15 g,89%); TLC [ethyl acetate]: R_(f)=0.06; melting point=261-262° C.(decomposition).

EXAMPLE 2.2

N-[Lysyl-alanyl]-batracyline, di-trifluoroacetate

2.2.a) N-[Nα,Nε-Di-(tert-butoxycarbonyl)-lysyl-alanyl]-batracyline

N,N-Di-(tert-butoxycarbonyl)-lysine (2.1 g, 6 mmol) and²-isobutoxy-1-isobutoxy-carbonyl-1,2-dihydro-quinoline (2.4 ml, 8 mmol)are dissolved in 20 ml of methylene chloride. After the mixture has beenstirred at room temperature for 20 minutes, a solution of compound 2.1(1.6 g, 5 mmol) in dimethylformamide (40 ml) is added and the batch isstirred at room temperature for a further 16 hours. It is thenconcentrated in vacuo and the residue is purified by flashchromatography [petroleum ether/ethyl acetate 2:1→1:1→ethyl acetate].Yellow crystals (2.89 g, 89%) are obtained; TLC [ethyl acetate]:R_(f)=0.52; melting point=203-204° C.

2.2) N-[Lysyl-alanyl]-batracyline, di-trifluoroacetate

Anhydrous trifluoroacetic acid (10 ml) is added to a suspension of theabove compound (2.6 g, 4 mmol) in methylene chloride (25 ml) and theresulting solution is stirred at room temperature for 30 minutes. Afterconcentration in vacuo, the residue is crystallized by addition ofdiethyl ether (100 ml). The precipitate is filtered off and washedintensively with diethyl ether. Yellow crystals (2.68 g, 99%) areobtained; TLC [ethyl acetate]: R_(f)=0.05; melting point=144-146° C.(decomposition).

EXAMPLE 2.3

N-[D-Alanyl]-batracyline

2.3.a) N-[N-Benzyloxycarbonyl-D-alanyl]-batracyline

N-Benzyloxycarbonyl-D-alanine (3.9 g, 17.5 mmol) is reacted as describedin Example 2.1.a and the product is worked up. The resulting yellowcrystals (6.4 g, 80%) are separated off by filtration, the combinedfiltrates are concentrated in vacuo and the residue is purified by flashchromatography [petroleum ether/ethyl acetate 3:2→1:1]. A further 1.35 g(17%) are obtained; TLC [ethyl acetate]: R_(f)=0.45; melting point=256°C.; [α]²⁰=+75.1° (c=1.0/CH₂Cl₂)+0.5% CH₃OH).

2.3) N-[D-Alanyl]-batracyline

Compound 2.3.a (11.4 g, 25 mmol) is dissolved in a 33% strength solutionof hydrogen bromide in glacial acetic acid (100 ml). After 30 minutes atroom temperature, the batch is concentrated to 30 ml in vacuo and theconcentrate is then poured into saturated sodium bicarbonate solution(1000 ml), while stirring vigorously. Stirring is continued for 10minutes, the mixture is filtered and the residue is washed with water, alittle isopropanol and diethyl ether. The product is obtained in yellowcrystals (7.87 g, 98%); TLC [ethyl acetate]: R_(f)=0.06; meltingpoint=267° C. (decomposition).

EXAMPLE 2.4

N-[N^(α)-(tert-Butoxycarbonyl)-lysyl-D-alanyl]-batracyline

2.4.a)N-[N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracyline

N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysine(5.3 g, 11.3 mmol) and2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (4 ml, 14 mmol)are dissolved in 40 ml of methylene chloride. After the mixture has beenstirred at room temperature for 20 minutes, a solution of compound 2.3(3.2 g, 10 mmol) in dimethylformamide (80 ml) is added and the batch isstirred at room temperature for a further 16 hours. It is thenconcentrated in vacuo and the residue is suspended in methylene chloride(100 ml). The resulting suspension is topped up with diethyl ether (300ml). After filtration and washing of the material in the filter withdiethyl ether, yellow crystals (5.65 g, 65%) are obtained; TLC [ethylacetate]: R_(f)=0.45; melting point=186° C.

2.4) N-[N^(α)-(tert-Butoxycarbonyl)-lysyl-D-alanyl]-batracyline

The above compound (5.6 g, 7.3 mmol) is dissolved in dimethylformamide(50 ml). After addition of piperidine (50 ml), the mixture is stirred atroom temperature for 3 hours and then concentrated in vacuo and theresidue is purified by flash chromatography [methylenechloride/methanol/ammonia (25%) 15:3:0.1→15:5:0.1]. Yellow crystals (2.5g, 62%) are obtained; melting point=217° C. (decomposition).

EXAMPLE 2.5

N-[N^(α)-(Fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracyline,trifluoroacetate

2.5.a)N-[N^(α)-(Fluorenyl-9-methoxycarbonyl)-N^(ε)-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracyline

N^(α)-(Fluorenyl-9-methoxycarbonyl)-N^(ε)-(tert-butoxycarbonyl)-lysine(5.3 g, 11.3 mmol) is reacted as described in Example 2.4.a and theproduct is purified. Yellow crystals (7.0 g, 80%) are obtained; TLC[ethyl acetate]: R_(f)=0.51; melting point=223° C.

2.5) N-[N^(α)-(Fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracyline,trifluoroacetate

Compound 2.5.a (6.17 g, 8 mmol) is reacted as described in Example 2.2.After concentration in vacuo, the residue is reprecipitated frommethylene chloride/diethyl ether to give yellow crystals (6.08 g, 97%);TLC [ethyl acetate]: R_(f)=0.05; melting point=224° C. (decomposition).

EXAMPLE 2.6

N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracyline,trifluoroacetate

Compound 2.4.a (6.17 g, 8 mmol) is reacted as described in Example 2.2.After concentration in vacuo, the residue is reprecipitated frommethylene chloride/diethyl ether to give yellow crystals (5.97 g, 95%);TLC [ethyl acetate]: R_(f)=0.04; melting point=188° C. (decomposition).

EXAMPLE 2.7

N-[Lysyl-D-asparagyl]-batracyline, di-trifluoroacetate

2.7.a) N-[N-(Fluorenyl-9-methoxycarbonyl)-D-asparagyl-(β-tert-butylester)]-batracyline

N-(Fluorenyl-9-methoxycarbonyl)-D-asparagyl-(β-tert-butyl ester) (7.2 g,17.5 mmol) is reacted as described in Example 2.1.a. After concentrationin vacuo, the residue is taken up in methylene chloride (1000 ml) andthe mixture is washed with 1N hydrochloric acid (2×200 ml) and with 1Nsodium bicarbonate solution (1×200 ml). After drying over magnesiumsulphate (20 g), filtration, concentration to 100 ml and addition ofpetroleum ether, compound 2.7.a is obtained in the form of yellowcrystals (9.7 g, 86%); TLC [ethyl acetate]: R_(f)=0.71; meltingpoint=195° C.

2.7.b) N-[D-Asparagyl-(β-tert-butyl ester)]-batracyline

The above compound (6.4 g, 10 mmol) is dissolved in methylene chloride(100 ml). After addition of morpholine (50 ml), the mixture is stirredat room temperature for 5 hours and then concentrated in vacuo and theresidue is purified by flash chromatography [ethyl acetate/petroleumether 4:1→ethyl acetate→ethyl acetate/ethanol 10:1]. Yellow crystals(3.44 g, 82%) are obtained; TLC [ethyl acetate]: R_(f)=0.21; meltingpoint=209° C. (decomposition).

2.7.c)N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-D-asparagyl-(β-tert-butylester)-batracyline

Compound 2.7.b (2.1 g, 5 mmol) is reacted as described in Example 2.2.a.Yellow crystals (1.71 g, 46%) are obtained; TLC [ethyl acetate]:R_(f)=0.61; melting point=142° C.

2.7) N-[Lysyl-D-asparagyl]-batracyline, di-trifluoroacetate

Compound 2.7.c (1.65 g, 2.2 mmol) is reacted as described in Example 2.2and the product is purified. Yellow crystals (1.5 g, 95%) are obtained;TLC [methanol/acetic acid 10:1]: R_(f)=0.29; melting point=154-155° C.(decomposition).

EXAMPLE 2.8

N-[Lysyl-D-glutamyl]-batracyline, di-hydrobromide

2.8.a) N-[N-(tert-Butoxycarbonyl)-D-glutamyl-(β-benzylester)]-batracyline

N-(tert-Butoxycarbonyl)-D-glutamyl-(β-benzyl ester) (5.9 g, 17.5 mmol)is reacted as described in Example 2.1.a. After concentration in vacuo,the residue is purified by flash chromatography [petroleum ether/ethylacetate 1:1] to give yellow crystals (9.45 g, 95%); TLC [ethyl acetate]:R_(f)=0.61; [α]²⁰=+53.1° (c=1.0/CH₂Cl₂); melting point=159° C.

2.8.b) N-[D-Glutamyl-(β-benzyl ester)]-batracyline

Compound 2.8.a (9.1 g, 10 mmol) is dissolved in formic acid (100 ml) andthe solution is stirred at room temperature for 6 hours. Afterconcentration in vacuo, the residue is taken up in methanol (100 ml) andthe pH of the solution is increased to pH 8 by careful addition of 25%strength aqueous ammonia solution. After renewed concentration in vacuo,subsequent flash chromatography [ethyl acetate/ethanol 10:1] gives ayellow oil (4.2 g, 56%); TLC [ethyl acetate]: R_(f)=0.06.

2.8.c)N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-D-glutamyl-(β-benzylester)]-batracyline

The above compound (3.75 g, 8 mmol) is reacted as described in Example2.2.a and the product is purified. A yellow amorphous solid (2.26 g,35%) is obtained; TLC [ethyl acetate]: R_(f)=0.40; [α]²⁰=+32.1°(c=1.2/CH₂Cl₂).

2.8) N-[Lysyl-D-glutamyl]-batracyline, di-hydrobromide

Compound 2.8.c (2.0 g, 2.5 mmol) is dissolved in a 33% strength solutionof hydrogen bromide in glacial acetic acid (50 ml). After 1 hour at roomtemperature, the batch is concentrated in vacuo and the residue iswashed thoroughly with diethyl ether. The product is obtained inyellow-red crystals (1.63 g, 98%); melting point=207-209° C.(decomposition).

EXAMPLE 2.9

N-[Lysyl-glycyl]-batracyline, di-trifluoroacetate

2.9.a) N-[N-(tert-Butoxycarbonyl)-glycyl]-batracyline

N-(tert-Butoxycarbonyl)-glycine (3.07 g, 17.5 mmol) is reacted asdescribed in Example 2.1.a. After 3 days at room temperature, themixture is concentrated in vacuo and the residue is taken up in ethanol(200 ml). After the mixture has been stirred under reflux for 30 minutesand filtered, after cooling, the target compound is obtained in the formof yellow crystals (4.73 g, 66%); TLC [ethyl acetate]: R_(f)=0.44;melting point=279° C. (decomposition).

2.9.b) N-Glycyl-batracyline, hydrochloride

The above compound (4.1 g, 10 mmol) is dissolved in methylene chloride(1200 ml), while heating in an ultrasonic bath. After addition ofhydrogen chloride in diethyl ether (100 ml), the mixture is stirred atroom temperature for 30 minutes and concentrated in vacuo, and ethanol(200 ml) is added to the residue. After the mixture has been stirredunder reflux for 10 minutes and filtered, after cooling, the product isobtained in the form of yellow crystals (3.21 g, 94%); melting point297-299° C. (decomposition).

2.9.c) N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-glycyl]-batracyline

N,N-Di-(tert-butoxycarbonyl)-lysine (2.1 g, 6 mmol) and2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (2.4 ml, 8 mmol)are dissolved in 20 ml of methylene chloride. After the mixture has beenstirred at room temperature for 20 minutes, a solution of compound 2.9.b(1.71 g, 5 mmol), ethyldiisopropylamine (0.86 ml, 5mmol) anddimethylformamide (40 ml) is added and the batch is stirred at roomtemperature for a further 16 hours. It is then concentrated in vacuo andthe residue is purified by flash chromatography [ethyl acetate/petroleumether 1:1→ethyl acetate]. Yellow crystals (1.69 g, 53%) are obtained;TLC [ethyl acetate]: R_(f)=0.31; melting point=211 ° C. (decomposition).

2.9) N-[Lysyl-glycyl]-batracyline, di-trifluoroacetate

Compound 2.9.c (1.4 g, 2.2 mmol) is reacted as described in Example 2.2and the product is purified. Yellow crystals (1.33 g, 91%) are obtained;melting point=153° C. (decomposition).

EXAMPLE 2.10

N-[Lysyl-seryl]-batracyline, di-trifluoroacetate

2.10.a) N-[N-(tert-Butoxycarbonyl)-seryl]-batracyline

N-(tert-Butoxycarbonyl)-serine (3.6 g, 17.5 mmol) is reacted asdescribed in Example 2.1.a. After 48 hours, the mixture is concentratedto 100 ml in vacuo and 21 of methylene chloride are added to theconcentrate. The resulting solution is washed with water (1×500 ml),with 0.5 N hydrochloric acid (2×250 ml) and with saturated sodiumbicarbonate solution (1×250 ml). Drying over magnesium sulphate (50 g),distilling off the solvent in vacuo and flash chromatography [petroleumether/ethyl acetate 1:1] of the residue gives compound 2.10.a (4.6 g,64%) in the form of yellow crystals; TLC [ethyl acetate/acetic acid100:1]: R_(f)=0.38; melting point=219° C. (decomposition); [α]²⁰=−61.0°(c=0.5/CH₂Cl₂+0.5% CH₃OH).

2.10.b) N-Seryl-batracyline, hydrochloride

Concentrated hydrochloric acid (10 ml) is added to a suspension of theabove compound (4.6 g, 10.4 mmol) in dioxane (70 ml), while stirring,and the mixture is then stirred vigorously at room temperature for 1hour. It is subsequently concentrated in vacuo and the residue is driedunder an oil pump vacuum for 2 hours. After addition of ethanol (100ml), the mixture is boiled under reflux for 15 minutes. Cooling andfiltration with suction give orange crystals (1.97 g, 96%); TLC [ethylacetate]: R_(f)=0.05; [α]²⁰=+51.8° (c=1.0/H₂OH); melting point>270° C.(decomposition).

2.10.c) N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-seryl]-batracyline

Compound 2.10.b (1.86 g, 5 mmol) is reacted as described in Example2.9.c. After flash chromatography [ethyl acetate/petroleum ether2:1→ethyl acetate], yellow crystals (1.18 g, 36%) are obtained; TLC[ethyl acetate/acetic acid 100:1]: R_(f)=0.24; melting point=188° C.(decomposition); [α]²⁰=−13.1° (c=0.5/CH₂Cl₂+0.5% CH₃OH).

2.10) N-[Lysyl-seryl]-batracyline, di-trifluoroacetate

Compound 2.10.c (1.0 g, 1.5 mmol) is reacted as described in Example 2.2and the product is purified. Yellow crystals (1.0 g, 96%) are obtained;TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.10;melting point=188-190° C. (decomposition).

EXAMPLE 2.11

N-[Lysyl-D-seryl]-batracyline, di-hydrobromide

2.11.a)N-[N-(Fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-seryl]-batracyline

N-(Fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-serine (6.7 g, 17.5mmol) is reacted as described in Example 2.1.a. Concentration in vacuoand flash chromatography [petroleum ether/ethyl acetate 1:1] give thecompound 2.11.a (5.64 g, 52%) in the form of yellow crystals; TLC[methylene chloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.87;melting point=225-226° C. (decomposition).

2.11.b) N-[O-(tert-Butyl)-D-seryl]-batracyline

The above compound (2.89 g, 4.7 mmol) is reacted as described in Example2.7.b. Flash chromatography [petroleum ether/ethyl acetate 3:2→ethylacetate] gives the product as yellow crystals (1.15 g, 62%); TLC [ethylacetate]: R_(f)=0.11; melting point=197° C.

2.11.c)N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-O-(tert-butyl)-D-seryl]-batracyline

The above compound (1.1 g, 2.8 mmol) is reacted as described in Example2.2.a. Yellow crystals (1.94 g, 96%) are obtained; TLC [ethyl acetate]:R_(f)=0.59; melting point=208° C.

2.11.d) N-[Lysyl-O-(tert-butyl)-D-seryl]-batracyline,di-trifluoroacetate

Compound 2.11.c (1.08 g, 1.5 mmol) is reacted as described in Example2.2 and the product is purified. Yellow crystals (1.1 g, 98%) areobtained; TLC [methanol/acetic acid 10:1]: R_(f)=0.30; meltingpoint=128° C.

2.11) N-[Lysyl-D-seryl]-batracyline, di-hydrobromide

Compound 2.11.d (1.05 g, 1.4 mmol) is reacted as described in Example2.8 and the product is purified. Yellow-red crystals (846 mg, 96%) areobtained; melting point=247-248° C.

EXAMPLE 2.12

N-[Lysyl-D-threonyl]-batracyline, di-hydrobromide

2.12.a)N-[N-(Fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-threonyl]-batracyline

N-(Fluorenyl-9-methoxycarbonyl)-O-)tert-butyl)-D-threonine (6.96 g, 17.5mmol) is reacted as described in Example 2.1.a. Concentration in vacuoand flash chromatography [petroleum ether/ethyl acetate 1:1] give thecompound 2.12.a (7.45 g, 68%) in the form of yellow crystals; TLC [ethylacetate]: R_(f)=0.63; melting point=225-226° C.

2.12.b) N-1O-(tert-Butyl)-D-threonyl]-batracyline

Compound 2.12.a (3.8 g, 6 mmol) is reacted as described in Example2.7.b. After concentration in vacuo, the product is obtained as yellowcrystals (1.9 g, 78%); TLC [ethyl acetate]: R_(f)=0.21; meltingpoint=110-111° C.

2.12.c)N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-O-(tert-butyl)-D-threonyl]-batracyline

Compound 2.12.b (1.8 g, 4.5 mmol) is reacted as described in Example2.2.a. Yellow crystals (2.6 g, 79%) are obtained; TLC [ethyl acetate]:R_(f)=0.59; melting point=112° C.

2.12.d) N-[Lysyl-O-(tert-butyl)-D-threonyl]-batracyline,di-trifluoroacetate

The above compound (2.5 g, 3.4 mmol) is reacted as described in Example2.2 and the product is purified. Yellow crystals (2.5 g, 96%) areobtained; TLC [methanol/acetic acid 10:1]: R_(f)=0.30; meltingpoint=142° C. (decomposition).

2.12) N-[Lysyl-D-threonyl]-batracyline, di-hydrobromide

Compound 2.12.d (2.29 g, 3 mmol) is reacted as described in Example 2.8and the product is purified. Yellow-red crystals (1.86 g, 97%) areobtained; melting point=232° C. (decomposition).

EXAMPLE 2.13

N-[Lysyl-D-alanyl]-batracyline, di-trifluoroacetate

2.13.a)N-[N^(α),N^(ε)-Bis-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracyline

6 g (17.3 mmol) of N_(α),N^(ε)-bis-(tert-butoxycarbonyl)-lysine aredissolved in 75 ml of dimethylformamide, and 3 g (26 mmol) ofN-hydroxysuccinimide and 4.29 g (20.8 mmol) ofN,N′-dicyclohexylcarbodiimide are added at 0° C. After 3 hours, the ureaformed is filtered off, 5 g (15.6 mmol) of N-[D-alanyl]-batracyline(Example 2.3) are added to the filtrate and the mixture is stirred at20° C. for 16 hours. Residual urea is filtered off and discarded. Thefiltrate is concentrated, the residue is stirred with methanol and themixture is filtered. The filtrate is concentrated again and the residueis treated again with methanol. The mixture is again filtered and thefilter residues are combined. They are dissolved in methylenechloride/methanol 10:1 and the product is precipitated with ether. 8.22g (81%) of the crystalline target product are obtained.

2.13) N-[Lysyl-D-alanyl]-batracyline, di-trifluoroacetate

Preparation from 8.2 g of compound 2.13.a analogously to Example 2.2.Yield: 7.58 g (89%)

EXAMPLE 2.14

N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline,trifluoroacetate

2.14.a)N-[N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline

Preparation analogously to Example 2.4.a fromN^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysineand batracyline. Yield: 78%

2.14) N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline,trifluoroacetate

Preparation analogously to Example 2.5 from compound 2.14.a. Yield: 90%

EXAMPLE 2.15

N-[Lysyl-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline,di-trifluoroacetate

2.15.a)N-[N^(α),N^(ε)-Di-(tert-butoxycarbonyl)-lysyl-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline

3240 mg (4.54 mmol) of compound 2.14 are dissolved in 50 ml ofdimethylformamide, and 2550 mg (5.45 mmol) ofN^(α),N^(ε)-di-(tert-butoxycarbonyl)-lysine p-nitrophenyl ester and 938μl of ethyldiisopropylamine are added. The mixture is stirred at 20° C.for. 16 hours and concentrated and the residue is initially stirred withether. The mixture is filtered and the filter residue is stirred again,with methanol/ether 1:1. 3881 mg (92%) of the target product areobtained in this way after filtration with suction and drying.

2.15) N-[Lysyl-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline,di-trifluoroacetate

Deblocking of compound 2.15.a with anhydrous trifluoroaceticacid/methylene chloride 1:1 analogously to Example 2.2. Yield: 95% [TLC:methylene chloride/methanol/ammonia (17%) 15:6:0.6 R_(f)=0.08]

EXAMPLE 2.16

N-[Lysyl-N^(β)-(fluorenyl-9-methoxycarbonyl)-α,β-diaminopropionyl]-batracyline,di-trifluoroacetate

This peptide conjugate was prepared analogously to Examples 2.14 and2.15 via 4 stages from batracyline andN^(α)-(tert-butoxycarbonyl)-N^(β)-(fluorenyl-9-methoxycarbonyl)-diaminopropionicacid. [TLC: methylene chloride/methanol/glacial acetic acid 5:1:0.2R_(f)=0.15]

EXAMPLE 2.17

N-[Lysyl]-batracyline

Splitting off of Fmoc analogously to Example 2.4 fromN-[N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracylinetrifluoroacetate (Example 2.14). Yield: 65%

EXAMPLE 2.18

N-[Seryl-D-alanyl]-batracyline, trifluoroacetate

2.18.a) N-[N-(tert-Butoxycarbonyl)-seryl-D-alanyl]-batracyline

Preparation analogously to Example 2.13.a fromN-(tert-butoxycarbonyl)-serine and N-[D-alanyl]-batracyline (Example2.3). Yield: 77%

2.18) N-[Seryl-D-alanyl]-batracyline, trifluoroacetate

Preparation analogously to Example 2.2 from compound 2.18.a. Yield: 98%

EXAMPLE 2.19

N-[D-Alanyl-D-alanyl]-batracyline, trifluoroacetate

Preparation via 2 stages analogously Example 2.18.

EXAMPLE 2.20

M-[Glutamyl-D-alanyl]-batracyline

Preparation via 2 stages analogously to Example 2.18 starting fromN-tert-butoxycarbonyl-glutamyl-δ-tert-butyl ester andN-[D-alanyl]-batracyline (Example 2.3). After splitting off of the Boc,the mixture is concentrated, the residue is taken up in water, the pH isbrought to 7 with 0.1 N sodium hydroxide solution and the betaine isfiltered off with suction.

EXAMPLES 3.1-3.34

General Formula

EXAMPLE 3.1

N-{N^(ε)-[O-(β-L-Fucosyl)-4-hydroxy-phenylaminothio-carbonyl]-lysyl-D-alanyl}-batracyline

3.1.a)N-{N^(α)-(tert-Butoxycarbonyl)-N^(ε)-[O-(β-L-fucosyl)-4-hydroxy-phenylaminothiocarbonyl]-lysyl-D-alanyl}-batracyline

Thiophosgene (34 μl, 0.44 mmol) is added to 55 mg (0.22 mmol) ofp-aminophenyl β-L-fucoside in 10 ml of dioxane/water 1:1, whilestirring. After 10 minutes, the mixture is concentrated in vacuo and theresidue is dried under a high vacuum for 1 hour. The isothiocyanateobtained is then coupled in absolute dimethylformamide with 109 mg (0.21mmol) of N-[N^(α)-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracyline(Example 2.4) in the presence of 115 μl of ethyldiisopropylamine. Afterthe crude product has been precipitated twice from methanol/isopropanol,132 mg (75%) of the target product are obtained. [TLC: methylenechloride/methanol 9:1 R_(f)=0.151.

3.1)N-{N^(ε)-O-(β-L-Fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

127 mg (0.15 mmol) of the compound from Example 3.1.a are stirred in 10ml of methylene chloride with 6 ml of anhydrous trifluoroacetic acid at0° C. for 2 hours. The mixture is concentrated, the residue issubsequently distilled three times with 5 ml of methylene chloride andthe product is chromatographed with methylene chloride/methanol/ammonia(17%) 15:2:0.2. After subsequent freeze drying, 80 mg (71%) of thetarget product are obtained: [TLC: methylene chloride/methanol/ammonia(17% 15:4:0.4 R_(f)=0.3].

Analogously to Example 3.1, the following glycoconjugates are preparedfrom the partly protected peptide conjugate in Example 2.4 or from theisomeric N-[N^(α)-(tert-butoxycarbonyl)-lysyl-alanyl]-batracyline, whichis to be prepared analogously:

EXAMPLE 3.2

N-{N^(ε)-O-(2-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate from Example 1.1

Flash chromatography purification of the intermediate stage withmethylene chloride/methanol 95:5 and of the final stage with methylenechloride/methanol/ammonia (17%) 15:2:0.2. Yield: 55% [TLC: methylenechloride/methanol/glacial acetic acid 5:1:0.2 R_(f)=0.4].

EXAMPLE 3.3

N-{N^(ε)-[O-(2-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracyline

Educt:

carbohydrate from Example 1.1

Purification of the into mediate stage by precipitation from methylenechloride/methanol 1:1 with ether and flash chromatography purificationof the final stage with methylene chloride/methanol/ammonia (17%)15:2:0.2. Yield: 65% [TLC: methylene chloride/methanol/ammonia (17%)15:2:0.2 R_(f)=0.21].

EXAMPLE 3.4

N-{N^(ε)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.2

Flash chromatography purification of the intermediate stage withmethylene chloride/methanol 97.5:2.5 and precipitation of the finalstage from methanol with ether; yield: 59% [TLC: methylenechloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.19].

EXAMPLE 3.5

N-{N^(ε)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracyline

Educt:

carbohydrate from Example 1.2

Purification of the intermediate stage by precipitation from methylenechloride/methanol 1:1 with ether and flash chromatography purificationof the final stage with methylene chloride/methanol/ammonia (17%)15:2:0.2. Yield: 36% [TLC: methylene chloride/methanol/ammonia (17%)15:4:0.5 R_(f)=0.57].

EXAMPLE 3.6

N-{N^(ε)-[O-(3-O-Methyl-α-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate from Example 1.3

Purification of the intermediate stage by precipitation from methanolwith ether and flash chromatography purification of the final stage withmethylene chloride/methanol/ammonia (17%) 15:2:0.2. Yield: 44% [TLC:methylene chloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.15].

EXAMPLE 3.7

N-{N^(ε)-[O-(3-Deoxy-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.6

Flash chromatography purification of the intermediate stage withmethylene chloride/methanol 95:5 and precipitation of the final stagefrom methanol with ether. Yield: 35% [TLC: acetonitrile/water/glacialacetic acid 5:1:0.2 R_(f)=0.42].

EXAMPLE 3.8

N-{N^(ε)-[O-(3,4-Epoxy-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.8

Flash chromatography purification of the intermediate stage withmethylene chloride/methanol 95:5. Several precipitations of the finalstage from methanol with ether and subsequent stirring with ethylacetate. [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.49].

EXAMPLE 3.9

N-{N^(ε)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Educt:

carbohydrate from Example 1.10

Purification of the intermediate stage by stirring with methanol andcompletion of the precipitation with ether. Flash chromatographypurification of the final stage with methylene chloride/methanol/ammonia(17%) 15:3:0.3; later in the same system with 15:6:0.6. Thecorresponding fractions are concentrated, the residue is taken up inwater and the pH is brought to 7 with 0.1N sodium hydroxide solution.The mixture is filtered with suction, the filter residue is taken up indimethylformamide/water 1:3 and one equivalent of a 0.1N sodiumhydroxide solution is added. The mixture is concentrated and the sodiumsalt is taken up in water and lyophilized. Yield: 43%. [TLC: methylenechloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.15].

EXAMPLE 3.10

N-{N^(ε)-[O-(3-O-Carbamoylmethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Educt:

carbohydrate from Example 1.18

Purification of the intermediate stage by stirring with methanol andcompletion of the precipitation with ether. Flash chromatographypurification of the final stage with methylene chloride/methanol/ammonia(17%) 15:2:0.2. [TLC: methylene chloride/methanol/ammonia (17%) 15:3:0.3R_(f)=0.38]; melting point: 190° C. (decomposition).

EXAMPLE 3.11

N-{N^(ε)-[O-(4-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,acetate

Educt:

carbohydrate from Example 1.4

Flash chromatography purification of the intermediates stage withmethylene chloride/methanol 95:5 and of the final stage with methylenechloride/methanol/ammonia (17%) 15:2:0.2. After the concentration, oneequivalent of glacial acetic acid and 10 ml of water are added to theresidue and the mixture is lyophilized. Yield: 52% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.43]. FAB-MS:m/z=760=M+1.

EXAMPLE 3.12

N-{N^(ε)-[O-(α-D-Glucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

p-aminophenyl α-D-glucoside

Purification of the intermediate stage and of the final stage bystirring with methanol and completion of the precipitation with ether.Yield: 83% [TLC: methylene chloride/methanol/ammonia (17%) 15:8:0.8R_(f)=0.48].

EXAMPLE 3.13

N-{N^(ε)-[O-(α-D-Glucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracyline,trifluoroacetate

Educt:

p-aminophenyl α-D-glucoside

Analogous preparation to that of the isomer in Example 3.12

EXAMPLE 3.14

N-{N^(ε)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

3.14.a)N-{N^(α)-(tert-Butoxycarbonyl)-N^(ε)-[O-(3-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Thiophosgene (33.5 ml, 0.44 mmol) is added to a solution of compound1.25 (62.8 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml), while stirring.After 10 minutes, the mixture is concentrated in vacuo and the residueis dried under an oil pump vacuum for 1 hour. The isothiocyanateobtained is dissolved in absolute dimethylformamide (10 ml), andcompound 2.4 (109.7 mg, 0.2 mmol) and ethyldiisopropylamine (0.5 ml) areadded. The mixture is stirred at room temperature for 16 hours and thenconcentrated in vacuo and the residue is purified by flashchromatography [methylene chloride/methanol 20:1]. Yellow crystals(108.3 mg, 62 %) are obtained; TLC [methylene chloride/methanol 5:1]:R_(f)=0.42]; melting point=194-195° C. (decomposition).

3.14)N-{N^(ε)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

The tert-butoxycarbonyl group is split off from compound 3.14.a (105.1mg, 0.12 mmol) as described in Example 2.2. After concentration in vacuoand reprecipitation from methanol/diethyl ether, yellow crystals (57.4mg, 54%) are obtained; TLC [methylene chloride/methanol 5:1]:R_(f)=0.16; melting point=188-189° C. (decomposition).

The following glycoconjugates are prepared analogously to Example 3.14.aand 3.14 from peptide conjugate 2.4 (in each case 109.7 mg, 0.2 mmol):

EXAMPLE 3.15

N-{N^(ε)-[O-(β-D-Galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.23 (59.7 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1] gives yellow crystals (79.5 mg, 46%);TLC [methanol]: R_(f)=0.74; melting point=182° C.

Purification of the end product as described in Example 3.14 givesyellow crystals (77.1 mg, 44%); TLC [methanol]: R_(f)=0.27; meltingpoint=191-192° C. (decomposition).

EXAMPLE 3.16

N-{N^(ε)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.31 (79 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 30:1→20:1] gives yellow crystals (150.7 mg,85%); TLC [methylene chloride/methanol 10:1]: R_(f)=0.35; meltingpoint=197-199° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (137 mg, 76%); TLC [methylene chloride/methanol 10:1]:R_(f)=0.13; melting point=184-186° C. (decomposition).

EXAMPLE 3.17

N-{N^(ε)-[O-(3-O-Methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.42 (75.5 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 30:1→25:1] gives yellow crystals (124.1 mg,66%); TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.50; melting point=165° C.

Purification of the end product as described in Example 3.14 givesyellow crystals (107.8 mg, 57%); TLC [methylene chloride/methanol 4:1]:R_(f)=0.53; melting point=183° C. (decomposition).

EXAMPLE 3.18

N-{N^(ε)-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

compound 1.43 (77.3 mg, 0.22 mmol)

Purification of the intermediate stage by reprecipitation fromethanol/diethyl ether gives the sodium salt as yellow crystals (172 mg,91%); TLC [methanol]: R_(f)=0.71; melting point=225-228° C.

Purification of the end product as described in Example 3.14 givesyellow crystals (136.5 mg, 73%); TLC [methanol]: R_(f)=0.12; meltingpoint=217-220° C. (decomposition).

EXAMPLE 3.19

N-{N^(ε)-[-(3-O-Carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.44 (72.2 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1] gives yellow crystal (137.7 mg, 75%);TLC [methylene chloride/methanol 4:1]: R_(f)=0.41; meltingpoint=198-201° C. (decomposition).

Purification of the end product as in Example 3.14 gives yellow crystals(140.2 mg, 75%); TLC [methylene chloride/methanol 4:1]: R_(f)=0.16;melting point=188-190° C. (decomposition).

EXAMPLE 3.20

N-{N^(ε)-[O-(3-O-(N-Methyl-carbamoylmethyl)-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.45 (75.3 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol/ammonia (25%) 7:1:0.1] gives yellowcrystals (158.4 mg, 85%); TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.25; melting point=161-163° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (132.5 mg, 70%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.10; meltingpoint=191-193° C. (decomposition).

EXAMPLE 3.21

N-{N^(ε)-[O-(3-O-(N-Propyl-carbamoylmethyl)-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.46 (81.5 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol/ammonia (25%) 8:1:0.1] gives yellowcrystals (153.1 mg, 80%); TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.33; melting point=187° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (154.9 mg, 79%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.21; meltingpoint=179° C.

EXAMPLE 3.22

N-{N^(ε)-[O-(3-O-(N-Butyl-carbamoylmethyl)-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.47 (84.6 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 12:1] gives yellow crystals (132.7 mg,68%); TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.54; melting point=180-182° C.

Purification of the end product as described in Example 3.14 givesyellow crystals (115.2 mg, 58%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.30; meltingpoint=176° C.

EXAMPLE 3.23

N-{N^(ε-[O-()3,4-Dideoxy-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.52 (52.6 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 25:1] gives yellow crystals (127.4 mg,77%); TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]:R_(f)=0.60; melting point=166-167° C.

Purification of the end product as described in Example 3.14 givesyellow crystals (103.1 mg, 61%); TLC [methylenechloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.44; meltingpoint=173-175° C. (decomposition).

EXAMPLE 3.24

N-{N^(ε)-[O-(6-O-Acetyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.53 (78.2 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 25:1] gives yellow crystals (88.3 mg, 49%);TLC [methylene chloride/methanol 4:1]: R_(f)=0.61; meltingpoint=196-199° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (89.6 mg, 49%); TLC [methylene chloride/methanol 4:1]:R_(f)=0.31; melting point=186° C. (decomposition).

EXAMPLE 3.25

N-{N^(ε)-[O-(α-D-Mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.39 (59.7 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1] gives yellow crystals (101.7 mg,59%); TLC [methanol]: R_(f)=0.79; melting point=180° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (103.1 mg, 59%); TLC [methanol]: R_(f)=0.34; meltingpoint=177-178° C. (decomposition).

EXAMPLE 3.26

N-{N^(ε)[O-(3-O-Methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.40 (62.8 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 20:1] gives yellow crystals (56.6 mg, 32%);TLC [methylene chloride/methanol 5:1]: R_(f)=0.38; meltingpoint=191-192° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (46.6 mg, 26%); TLC [methylene chloride/methanol 5:1]:R_(f)=0.13; melting point=190-191 ° C. (decomposition).

EXAMPLE 3.27

N-{N^(ε)-[O-(2,3-Di-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.41 (66 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 25:1] gives yellow crystals (77.8 mg, 44%);TLC [methylene chloride/methanol 4:1]: R_(f)=0.65; meltingpoint=182-183° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (66.1 mg, 37%); TLC [methylene chloride/methanol 4:1]:R_(f)=0.40; melting point=181° C.

EXAMPLE 3.28

N-{N^(ε)-[O-(3-O-Methoxycarbonylmethyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.48 (75.5 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 18:1] gives yellow crystals (62.1 mg, 33%);TLC [methylene chloride/methanol/ammonia (25%) 15:3:0.2]: R_(f)=0.66;melting point=165° C.

Purification of the end product as described in Example 3.14 givesyellow crystals (57.6 mg, 30%); TLC [methylene chloride/methanol/ammonia(25%) 15:3:0.2]: R_(f)=0.43; melting point=183-184° C.

EXAMPLE 3.29

N-{N^(ε)-[O-(3-O-Carboxymethyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.49 (77.3 mg, 0.22 mmol)

Purification of the intermediate stage by reprecipitation fromethanol/diethyl ether gives the sodium salt as yellow crystals (173.4mg, 92%); TLC [methanol]: R_(f)=0.57; melting point=201-205° C.(decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (171.7 mg, 92%); TLC [methanol]: R_(f)=0.29; meltingpoint=196-198° C. (decomposition).

EXAMPLE 3.30

N-{N^(ε)-[O-(3-O-Carbamoylmethyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate 1.50 (72.2 mg, 0.22 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1] gives yellow crystals (106.6 mg,58%); TLC [methylene chloride/methanol 4:1]: R_(f)=0.34; meltingpoint=192-194° C. (decomposition).

Purification of the end product as described in Example 3.14 givesyellow crystals (107.7 mg, 58%); TLC [methylene chloride/methanol 4:1]:R_(f)=0.13; melting point=186-187° C. (decomposition).

EXAMPLE 3.31

N-{N^(ε)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

3.31.a)N-{N^(α)-(Fluorenyl-9-methoxycarbonyl)-N^(ε)-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Thiophosgene (33.5 ml, 0.44 mmol) is added to a solution of compound1.31 (79 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml). After 10 minutes,the mixture is concentrated in vacuo and the residue is dried under anoil pump vacuum for 1 hour. The isothiocyanate obtained is dissolved inabsolute dimethylformamide (10 ml), and compound 2.5 (157 mg, 0.2 mmol)and ethyldiisopropylamine (0.5 ml) are added. The mixture is stirred atroom temperature for 16 hours and then concentrated in vacuo and theresidue is taken up in methylene chloride/methanol 1:1. The product isprecipitated by addition of diethyl ether and washed with a littleice-cold methanol. Yellow crystals (191 mg, 94%) are obtained; TLC[methylene chloride/methanol 10:1]: R_(f)=0.35; melting point=203° C.(decomposition).

3.31)N-{N^(ε)-[-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

The fluorenyl-9-methoxycarbonyl group is split off from compound 3.31.a(182.2 mg, 0.18 mmol) as described in Example 2.4. After concentrationin vacuo and dissolving in methanol/methylene chloride 1:1, the productis precipitated by addition of diethyl ether. Yellow crystals (127.1 mg,89%) are obtained; TLC [methanol]: R_(f)=0.46; melting point=158° C.

The following glycoconjugates are prepared analogously to Examples3.31.a and 3.31 from peptide conjugate 2.5 (in each case 157 mg, 0.2mmol):

EXAMPLE 3.32

N-{N^(ε)-[O-(3-O-(Piperidyl-N)-carbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.42 (75.5 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 3.31.a;yellow crystals (191.2 mg, 91%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.28; melting point=208° C.(decomposition).

Purification of the end product as described in Example 3.31 givesyellow crystals (155.8 mg, 88%); TLC [methanol]: R_(f)=0.47; meltingpoint=120° C. (decomposition).

EXAMPLE 3.33

N-{N^(ε)-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Educt:

carbohydrate 1.43 (77.3 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 3.31.a;yellow crystals (192 mg, 90%) are obtained; TLC [ethanol/methanol 1:1]:R_(f)=0.05; melting point=211-213° C. (decomposition).

Purification of the end product as described in Example 3.31 givesyellow crystals (110.6 mg, 66%); TLC [methanol]: R_(f)=0.33; meltingpoint=233-235° C. (decomposition).

EXAMPLE 3.34

N-{N^(ε)-[O-(3-O-Carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.44 (72.2 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 3.31.a;yellow crystals (159.2 mg, 76%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.04; melting point=177° C.(decomposition).

Purification of the end product as described in Example 3.31 givesyellow crystals (125.1 mg, 76%); TLC [methanol]: R_(f)=0.48; meltingpoint=106° C. (decomposition).

EXAMPLES 4.1-4.12

General Formula

EXAMPLE 4.1

4.1.a)N-{N^(α)-[O-(β-L-Fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl}-batracyline

140 mg (0.55 mmol) of p-aminophenyl β-L-fucoside are first convertedinto the isothiocyanate in accordance with the instructions in Example3.1.a and the product is then coupled with 430 mg (0.55 mmol) ofN-[N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracylinetrifluoroacetate (Example 2.5) in the presence of 375 μl ofethyldiisopropylamine. After precipitation from methanol/methylenechloride, the crude product is purified by flash chromatography(acetonitrile/water 10:1). After the residue has been stirred withether, 358 mg (67%) of the target product are obtained. [TLC:acetonitrile/water 10:1 R_(f)=0.48].

4.1)N-{Nα-[O-(β-L-Fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

356 mg (0.37 mmol) of the compound from Example 4.1.a are dissolved in10 ml of dimethylformamide and 5 ml of piperidine and the solution isstirred at 20° C. for 1 hour. It is concentrated and the residue ischromatographed with methylene chloride/methanol/ammonia (17%) 15:6:0.6.The target product is obtained in a 46% yield. [TLC: methylenechloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.11].

The following glycoconjugates are prepared analogously to Examples 4.1from the partly protected peptide conjugate 2.5:

EXAMPLE 4.2

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Educt:

carbohydrate from Example 1.10

Chromatographic purification of the intermediate stage with methylenechloride/methanol/ammonia (17%) 15:3:0.3; later in the same system15:4:0.5.

Purification of the end product at the betaine stage by stirring withwater; subsequent conversion into the sodium salt with 0.1N sodiumhydroxide solution and freeze drying from dioxane/water. Yield: 65%;melting point: 220° C.

EXAMPLE 4.3

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate from Example 1.2

Purification of the intermediate stage by precipitation with methylenechloride with ether; purification of the end product by severalprecipitations from dimethylformamide with ether. Yield: 88% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.28].

EXAMPLE 4.4

N-{N^(α)-[O-(4-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate from Example 1.4

Purification of the intermediate stage by several precipitations frommethylene chloride/methanol 1:1 with ether; column chromatographypurification of the end product [methylene chloride/methanol/ammonia(17%) 15:8:0.8], precipitation from methylene chloride/methanol 1:1 withether. Yield: 74% [TLC: methylene chloride/methanol/ammonia (17%)10:10:1 R_(f)=0.19].

EXAMPLE 4.5

N-{N^(α)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

4.5.a)N-{N^(α)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl-N^(ε)-(fluorenyl-9-methoxycarbonyl)]-lysyl-D-alanyl}-batracyline

Thiophosgene (33.5 ml, 0.44 mmol) is added to a solution of compound1.31 (79 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml). After 10 minutes,the mixture is concentrated in vacuo and the residue is dried under anoil pump vacuum for 1 hour. The isothiocyanate obtained is dissolved inabsolute dimethylformamide (10 ml), and compound 2.6 (157 mg, 0.2 mmol)and ethyldiisopropylamine (0.5 ml) are added. The mixture is stirred atroom temperature for 16 hours and then concentrated in vacuo. Severalreprecipitations of the residue from methylene chloride/methanol 1:1 bymeans of diethyl ether and final washing with a little ice-cold methanolgives yellow crystals (198 mg, 98%); TLC [methylene chloride/methanol10:1]: R_(f)=0.23; melting point=175° C.

4.5)N-{N^(α)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

The fluorenyl-9-methoxycarbonyl group is split off from compound 4.5.a(172.1 mg, 0.17 mmol) as described in Example 2.4. After concentrationin vacuo and dissolving in methanol/methylene chloride 1:1, the productis precipitated by addition of diethyl ether. Yellow crystals (114.5 mg,85%) are obtained; TLC [methylene chloride/methanol 1:1]: R_(f)=0.16;melting point=206° C. (decomposition).

The following glycoconjugates are prepared analogously to Example 4.5.aand 4.5 from peptide conjugate 2.6 (in each case 157 mg, 0.2 mmol):

EXAMPLE 4.6

N-{N^(α)-[O-(β-D-Galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.23 (59.7 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (185.8 mg, 94%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.09; melting point=182° C.(decomposition).

Purification of the end product as described in Example 4.5 gives yellowcrystals (134.3 mg, 88%); TLC [methylene chloride/methanol 1:1]:R_(f)=0.04; melting point=221° C. (decomposition).

EXAMPLE 4.7

N-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.25 (62.8 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (193.5 mg, 97%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.27; melting point=178° C.(decomposition).

Purification of the end product by flash chromatography [methylenechloride/methanol 2:1→1:1] gives yellow crystals (130.5 mg, 84%); TLC[methylene chloride/methanol 1:1]: R_(f)=0.09; melting point=206° C.(decomposition).

EXAMPLE 4.8

N-{N^(α)-[O-(3-O-Methoxycarbonylmethyl-[-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.42 (75.5 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (209.8 mg, 99%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.32; melting point=235° C.(decomposition).

Purification of the end product as described in Example 4.5 gives yellowcrystals (164.3 mg, 99%); TLC [methylene chloride/methanol 1:1]:R_(f)=0.05; melting point=217° C. (decomposition).

EXAMPLE 4.9

N-{N^(α)-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Educt:

carbohydrate 1.43 (77.3 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (210.3 mg, 99%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.02; melting point=185° C.

After purification of the product as described in Example 4.5, theresidue is suspended in water (10 ml), and 0.05 N sodium hydroxidesolution is added dropwise to the suspension, while stirring, until aclear solution forms (pH<10). Lyophilization of the filtered solutiongives a yellow amorphous solid (150.8 mg, 90%); TLC [methylenechloride/methanol 1:1]: R_(f)=0.04.

EXAMPLE 4.10

N-{N^(α)-[-(3-O-Carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.44 (72.2 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (152.7 mg, 73%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.11; melting point=229° C.(decomposition).

After purification of the product as described in Example 4.5, theresidue is suspended in water/dioxane 1:1 (20 ml). Lyophilization of thefiltered solution gives a yellow amorphous solid (98.4 mg, 61%); TLC[methylene chloride/methanol 1:1]: R_(f)=0.10; [α]²⁰=+44.9° (c=0.2/H₂O).

EXAMPLE 4.11

N-{N-[O-(α-D-Mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.39 (59.7 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (179.6 mg, 91%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.07; melting point=176° C.(decomposition).

Purification of the end product as described in Example 4.5 gives yellowcrystals (137.5 mg, 90%); TLC [methylene chloride/methanol 1:1]:R_(f)=0.09; melting point=213° C. (decomposition).

EXAMPLE 4.12

N-{N^(α)-[O-(3-O-Methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

carbohydrate 1.40 (62.8 mg, 0.22 mmol)

Purification of the intermediate stage as described in Example 4.5.a;yellow crystals (94.2 mg, 48%) are obtained; TLC [methylenechloride/methanol 10:1]: R_(f)=0.13; melting point=173° C.(decomposition).

Purification of the end product as described in Example 4.5 gives yellowcrystals (66.6 mg, 43%); TLC [methylene chloride/methanol 1:1]:R_(f)=0.07; melting point=215° C. (decomposition).

EXAMPLES 5.1-5.23

General Formula

EXAMPLE 5.1

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxyphenylaminothiocarbonyl]-N^(ε)-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxyphenylaminothiocarbonyl]-lysyl-D-alanyl}-batracyline

30 μl (0.18 mmol) of thiophosgene are added to 50 mg (0.16 mmol) ofp-aminophenyl 3-O-carboxymethyl-β-L-fucoside (Example 1.10) in 10 ml ofdioxane/water 1:1, while stirring. After 10 minutes, the mixture isconcentrated and the residue is dried under a high vacuum for 1 hour.The isothiocyanate obtained is then coupled in absolutedimethylformamide with 109 mg (0.144 mmol) of the conjugate from Example3.4 in the presence of 82 μl of ethyldiisopropylamine. The mixture isconcentrated and the residue is purified by flash chromatography[methylene chloride/methanol/ammonia (17%) 15:4:0.5]. The substanceobtained after concentration is lyophilized from water. Yield: 89 mg(56%). [TLC: methylene chloride/methanol/ammonia (17%) 15:6:0.6R_(f)=0.22].

The following conjugates with mixed substituents are preparedanalogously to Example 5.1:

EXAMPLE 5.2

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educts:

carbohydrate from Example 1.2, conjugate from Example 3.10

Purification by precipitation of the crude product from methanol withether. Yield: 78% [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.45].

EXAMPLE 5.3

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε-[)4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educts:

conjugate from Example 4.3, 4-hydroxy-aniline

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.21; yield: 60% [TLC: methylenechloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.31].

EXAMPLE 5.4

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educts:

conjugate from Example 4.2, 4-hydroxy-aniline

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:4:0.5]. The residue is then stirredwith methanol/ether. Yield: 55% [TLC: methylenechloride/methanol/ammonia (17%) 15:6:0.6 R_(f)=0.3].

EXAMPLE 5.5

N-{N^(α)-[O-(4-O-Methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educts:

conjugate from Example 4.4, 4-hydroxy-aniline

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]. The residue is thenprecipitated from methanol/methylene chloride with ether. Yield: 49%melting point: 128° C. [TLC: methylene chloride/methanol/ammonia (17%)15:2:0.2 R_(f)=0.26].

EXAMPLE 5.6

N-{N^(α)-[4-Hydroxyphenylamino-thiocarbonyl]-N^(ε)-[O-(4-O-methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educts:

conjugate from Example 3.11, 4-hydroxy-aniline

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]. The residue is thenlyophilized from water/dioxane. Yield: 50% [TLC: methylenechloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.291.

EXAMPLE 5.7

N-{N^(α)-[4-Hydroxyphenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educts:

conjugate from Example 3.4, 4-hydroxy-aniline

Precipitate the residue from methanol with ether. Yield: 76% [TLC:methylene chloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.26].

EXAMPLE 5.8

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[4-hydroxyethylamino-2-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxyphenylamino]triazin-6-yl]]-lysyl-D-alanyl}-batracyline

Educts:

conjugate from Example 8.10, carbohydrate from Example 1.2

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]. Yield: 9% FAB-MS:m/z=1165=M+1 [TLC: methylene chloride/methanol/ammonia (17%) 15:3:0.3R_(f)=0.27].

EXAMPLE 5.9

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxyphenylaminothiocarbonyl]-N^(α)-[acetyl]-lysyl-D-alanylbatracyline

Educts:

N-[N^(ε)-(acetyl)-lysyl-D-alanyl]-batracyline, carbohydrate from Example1.10

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2; later in the same system15:4:0.5]. The residue is then freeze dried from water. Yield: 46% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.31].

EXAMPLE 5.10

N-{N-[O-(4-O-Methyl-β-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[acetyl]-lysyl-D-alanyl}-batracyline

10 μl of acetic anhydride are added to 51 mg (0.067 mmol) of theconjugate from Example 4.4 in 5 ml of dimethylformamide and the mixtureis stirred at room temperature for 30 minutes. It is concentrated andthe residue is precipitated from methanol with ether. Yield: 46 mg (86%)[TLC: acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.6].

EXAMPLE 5.11

N-{N^(α)-[O-(β-L-Fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[succinyl]-lysyl-D-alanyl}-batracyline,sodium salt

3 mg of succinic anhydride are added to 20 mg (0.027 mmol) of theconjugate from Example 4.1 in 2 ml of dimethylformamide and the mixtureis stirred at room temperature for 6 hours. It is concentrated and theresidue is precipitated from methanol with ether. The residue is takenup in water, and 27 μl of a 0.1N sodium hydroxide solution are added.Yield: 20 mg (86%); [TLC: methylene chloride/methanol/glacial aceticacid 80:20:2 R_(f)=0.2].

EXAMPLE 5.12

N-{N^(α)-[O-(3-O-Methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-N^(ε)-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Carbohydrate 1.42 (37.7 mg, 0.11 mmol) is reacted with peptide conjugate3.31 (79 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and flash chromatography [methylenechloride/methanol 20:1→10:1], yellow crystals (52.9 mg, 45%) areobtained; TLC [methylene chloride/methanol 10:1]: R_(f)=0.14; meltingpoint=178° C. (decomposition).

EXAMPLE 5.13

N-{N^(α)-[-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylaminothiocarbonyl]-N^(ε)-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Carbohydrate 1.43 (38.6 mg, 0.11 mmol) is reacted with peptide conjugate3.31 (79 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and dissolving in methanol/methylene chloride1:1, the product is precipitated by addition of diethyl ether. Theresidue is suspended in water (10 ml), and 0.05 N sodium hydroxidesolution is added dropwise to the suspension, while stirring, until aclear solution forms (pH<10). Lyophilization of the filtered solutiongives a yellow amorphous solid (87.9 mg, 74%); TLC [ethanol]:R_(f)=0.17; [α]²⁰=+56.0° (c=0.1/H₂O).

EXAMPLE 5.14

N-{N^(α)-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Carbohydrate 1.44 (36.1 mg, 0.11 mmol) is reacted with peptide conjugate3.31 (79 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and dissolving in methanol/methylene chloride1:1, the product is precipitated by addition of diethyl ether. Yellowcrystals (45.9 mg, 39%) are obtained; TLC [ethanol]: R_(f)=0.38; meltingpoint=219° C. (decomposition).

EXAMPLE 5.15

N-{N^(α)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-(piperidyl-N)-carbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Carbohydrate 1.31 (39.5 mg, 0.11 mmol) is reacted with peptide conjugate3.32 (88.7 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and flash chromatography [methylenechloride/methanol 20:1→10:1], yellow crystals (38.8 mg, 32%) areobtained; TLC [methylene chloride/methanol 5:1]: R_(f)=0.79; meltingpoint=205° C. (decomposition).

EXAMPLE 5.16

N-{N^(α)-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-(piperidyl-N)-carbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Carbohydrate 1.43 (38.6 mg, 0.11 mmol) is reacted with peptide conjugate3.32 (88.7 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and dissolving in methanol/methylene chloride1:1, the product is precipitated by addition of diethyl ether. Theresidue is suspended in water (10 ml), and 0.05N sodium hydroxidesolution is added dropwise to the suspension, while stirring, until aclear solution forms (pH<10). Lyophilization of the filtered solutiongives a yellow amorphous solid (90.3 mg, 71%); TLC [ethanol]:R_(f)=0.05; [α]²⁰=+39.0° (c=0.1/H₂O).

EXAMPLE 5.17

N-{N^(α)-[O-(3-O-Carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-(piperidyl-N)-carbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl-lysyl-D-alanyl}-batracyline

Carbohydrate 1.44 (36.1 mg, 0.11 mmol) is reacted with peptide conjugate3.32 (88.7 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and dissolving in methanol/methylene chloride1:1, the product is precipitated by addition of diethyl ether. Yellowcrystals (44.8 mg, 36%) are obtained; TLC [ethanol]: R_(f)=0.06; meltingpoint=223° C. (decomposition).

EXAMPLE 5.18

N-{N-^(α)[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Carbohydrate 1.31 (39.5 mg, 0.11 mmol) is reacted with peptide conjugate3.33 (84.2 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo, methanol/methylene chloride 1:1 (20 ml) is addedto the residue and the product is precipitated by addition of diethylether. The residue is suspended in water (10 ml), and 0.05N sodiumhydroxide solution is added dropwise to the suspension, while stirring,until a clear solution forms (pH<10). Lyophilization of the filteredsolution gives a yellow amorphous solid (80.7 mg, 68%); TLC [ethanol]:R_(f)=0.09; [α]²⁰=+35.0° (c=0.1/H₂O).

EXAMPLE 5.19

N-{N^(α)-[O-(3-O-Methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Carbohydrate 1.42 (37.7 mg, 0.11 mmol) is reacted with peptide conjugate3.33 (84.2 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo, methanol/methylene chloride 1:1 (20 ml) is addedto the residue and the product is precipitated by addition of diethylether. The residue is suspended in water (10 ml), and 0.05N sodiumhydroxide solution is added dropwise to the suspension, while stirring,until a clear solution forms (pH<10). Lyophilization of the filteredsolution gives a yellow amorphous solid (87.5 mg, 71%); TLC [ethanol]:R_(f)=0.10; [α]²⁰=+24.6° (c=0.11/H₂O).

EXAMPLE 5.20

N-{N^(α)-[O-(3-O-Carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Carbohydrate 1.44 (36.1 mg, 0.11 mmol) is reacted with peptide conjugate3.33 (84.2 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo, methanol/methylene chloride 1:1 (20 ml) is addedto the residue and the product is precipitated by addition of diethylether. The residue is suspended in water (10 ml), and 0.05N sodiumhydroxide solution is added dropwise to the suspension, while stirring,until a clear solution forms (pH<10). Lyophilization of the filteredsolution gives a yellow amorphous solid (78.6 mg, 65%); TLC [ethanol]:R_(f)=0.11; [α]²⁰=−44.0° (c=0.13/H₂O).

EXAMPLE 5.21

N-{N^(α)-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Carbohydrate 1.31 (39.5 mg, 0.11 mmol) is reacted with peptide conjugate3.34 (81.9 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and flash chromatography[ethanol→ethanol/methanol 2:1], yellow crystals (17.4 mg, 15%) areobtained; TLC [ethanol]: R_(f)=0.20; melting point >290° C.(decomposition).

EXAMPLE 5.22

N-{N^(α)-[O-(3-O-Methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Carbohydrate 1.42 (37.7 mg, 0.11 mmol) is reacted with peptide conjugate3.34 (81.9 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo and dissolving in methanol/methylene chloride1:1, the product is precipitated by addition of diethyl ether. Yellowcrystals (72 mg, 60%) are obtained; TLC [ethanol]: R_(f)=0.21; meltingpoint 222° C. (decomposition).

EXAMPLE 5.23

N-{N^(α)-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,sodium salt

Carbohydrate 1.43 (38.6 mg, 0.11 mmol) is reacted with peptide conjugate3.34 (81.9 mg, 0.1 mmol) as described in Example 3.31.a. Afterconcentration in vacuo, and dissolving in methanol/methylene chloride1:1, the product is precipitated by addition of diethyl ether. Theresidue is suspended in water (10 ml), and 0.05N sodium hydroxidesolution is added dropwise to the suspension, while stirring, until aclear solution forms (pH<10). Lyophilization of the filtered solutiongives a yellow amorphous solid (78.2 mg, 65%); TLC [ethanol]:R_(f)=0.07; [α]²⁰=+33.0° (c=0.1/H₂O).

EXAMPLES 6.1-6.89

General Formula

EXAMPLE 6.1

N-{N^(α),N^(ε)-Bis-[O-(2-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Thiophosgene (30 μl, 0.4 mmol) is added to a solution of compound 1.1(50 mg, 0.19 ml) in dioxane/water 1:1 (10 ml), while stirring. After 10minutes, the mixture is concentrated in vacuo and the residue is driedunder an oil pump vacuum for 1 hour. The isothiocyanate obtained isdissolved in absolute dimethylformamide (10 ml), and compound 2.13 (61mg, 0.09 mmol) and ethyldiisopropylamine (0.5 ml) are added. The mixtureis stirred at room temperature for 16 hours and then concentrated invacuo and the residue is purified by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]. The residue is precipitatedfrom methanol with ether. 48 mg (50%) of the target product are obtainedas yellow crystals.

The following glycoconjugates are prepared analogously to Example 6.1from the peptide conjugate in Example 2.13 or the L-alanyl isomer(Example 2.2):

EXAMPLE 6.2

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

100 mg (0.38 mmol) of carbohydrate from Example 1.2

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:0.5:0.05; later 15:1:0.1 in the samesystem]; subsequent precipitation from methylenechloride/methanol/ether. Yield: 59%. Melting point 178° C.(decomposition) [TLC: acetonitrile/water 10:1 R_(f)=0.51].

EXAMPLE 6.3

N-{N^(α),N^(ε)-Bis-[O-(4-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

115 mg (0.44 mmol) of carbohydrate from Example 1.4

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]; subsequent precipitation frommethylene chloride/methanol (1:1)/ether. Yield: 58%. Melting point 176°C. (decomposition) [TLC: acetonitrile/water 10:1 R_(f)=0.52].

EXAMPLE 6.4

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracyline

Educt:

115 mg (0.44 mmol) of carbohydrate from Example 1.2

Purification by precipitation from methylene chloride/methanol (1:1)/ether. Yield: 93%. Melting point 192° C. (decomposition) [TLC:acetonitrile/water 10:1 R_(f)=0.46].

EXAMPLE 6.5

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-α-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

100 mg (0.38 mmol) of carbohydrate from Example 1.3

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:1:0.1]; precipitation from methylenechloride/methanol (1:1)/ether. Melting point: 178° C. (decomposition);FAB-MS: m/z=1071=M+1.

EXAMPLE 6.6

N-{N^(α),N^(ε)-Bis-[O-(3-O-n-propyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracyline

Educt:

38 mg (0.127 mmol) of carbohydrate from Example 1.5

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:1:0.1]; precipitation from methylenechloride/methanol (1:1)/ether. Yield: 42%, melting point: 167-170° C.(decomposition), [TLC: methylene chloride/methanol/ammonia (17%)R_(f)=0.34].

EXAMPLE 6.7

N-{N^(α),N^(ε)-Bis-[O-(3-O-n-propyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]lysyl-D-alanyl}-batracyline

Educt:

40 mg (0.167 mmol) of carbohydrate from Example 1.6

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]; precipitation frommethanol/ether. Yield: 81% [TLC: acetonitrile/water 10:1 R_(f)=0.46].

EXAMPLE 6.8

N-{N^(α,N)^(ε)-Bis-[O-(3,4-dideoxy-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

41 mg (0.183 mmol) of carbohydrate from Example 1.7

Purification by flash chromatography [methylene chloride/methanol 95:5];freeze drying from water/dioxane. Yield: 60% [TLC: methylenechloride/methanol 9:1 R_(f)=0.22].

EXAMPLE 6.9

N-{N^(α),N^(ε)-Bis-[O-(3-hydroxyethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

75 mg (0.25 mmol) of carbohydrate from Example 1.12

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2; later in the same system15:4:0.5]. Precipitation from methanol/ether. Yield: 66%; [TLC:acetonitrile/water 10:1 R_(f)=0.28].

EXAMPLE 6.10

N-{N^(α),N^(ε)-Bis-[O-(2-hydroxyethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

50 mg (0.167 mmol) of carbohydrate from Example 1.19

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:3:0.3]; precipitation frommethanol/ether; freeze drying from water/dioxane. Yield: 72%; [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.39].

EXAMPLE 6.11

N-{N^(α),N^(ε)-Bis-[O-(2-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,di-sodium salt

Educt:

50 mg (0.16 mmol) of carbohydrate from Example 1.13

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:8:0.8; later in the same system15:10:1]. Digest the residue with ether, subsequent freeze drying fromwater/dioxane. Conversion into the di-sodium salt with 2 equivalents ofa 0.1N sodium hydroxide solution, subsequently freeze drying from water.Yield: 49% [TLC: acetonitrile/water/glacial acetic acid 5:1:0.5R_(f)=0.38]. MS-FAB: FAB⁻; m/z=1157=M−2Na⁺+H⁺.

EXAMPLE 6.12

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,di-sodium salt

Educt:

200 mg (0.64 mmol) of carbohydrate from Example 1.10

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:4:0.5; later in the same system15:8:0.8; and finally 15:10:1]. Digest the residue with ether,subsequently freeze drying from water/dioxane 1:1. Conversion into thedi-sodium salt with 2 equivalents of a 0.1N sodium hydroxide solution,subsequently freeze drying from water. Yield: 59%; [TLC: methylenechloride/methanol/ammonia (17%) R_(f)=0.1].

EXAMPLE 6.13

N-[N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

60 mg (0.19 mmol) of carbohydrate from Example 1.18

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:3:0.3]. Precipitate the residue frommethylene chloride/methanol (1:1) with ether, filter off with suctionand subsequently freeze drying from water/dioxane 1:1. Yield: 36% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.461. Meltingpoint: 190° (decomposition).

EXAMPLE 6.14

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

100 mg (0.39 mmol) of p-aminophenyl β-L-fucoside

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2; later in the same system15:4:0.5]. Precipitate the residue from dimethylformamide with ether,filter off with suction. Yield: 48%; melting point: 195-198° C.

EXAMPLE 6.15

N-{N^(α),N^(ε)-Bis-[O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Educt:

158 mg (0.61 mmol) of carbohydrate from Example 1.21

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:3:0.3; later in the same system15:4:0.5]. Lyophilize the residue from water/dioxane. Yield: 87% [TLC:methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.25].

EXAMPLE 6.16

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,di-sodium salt

Educt:

200 mg (0.64 mmol) of carbohydrate from Example 1.22

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:4:0.5; later in the same system15:8:0.8; and finally 15:10:1]. Digest the residue with ether,subsequently freeze drying from water/dioxane 1:1.

The following glycoconjugates which contain a completely deblockedlysine structural unit on the amino end are prepared analogously toExample 6.1 from various peptide conjugates of batracyline:

EXAMPLE 6.17

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline,di-sodium salt

Educts:

32 mg (0.1 mmol) of carbohydrate from Example 1.10

32 mg (0.045 mmol) of N-[lysyl-glycyl]-batracyline, di-trifluoroacetate(Example 2.9)

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:8:0.8; later in the same system15:15:1.51. Precipitation from dimethylformamide/methanol (1:1) withether, subsequent freeze drying from water/dioxane. Conversion into thedi-sodium salt with 2 equivalents of a 0.1N sodium hydroxide solution,subsequently freeze drying from water. Yield: 25%; [TLC: methylenechloride/methanol/ammonia (17%) 15:8:0.8 R_(f)=0.19]. FAB-MS:m/z=1189=M+1.

EXAMPLE 6.18

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Educts:

60 mg (0.22 mmol) of carbohydrate from Example 1.2

66 mg (0.1 mmol) of N-[lysyl-glycyl]-batracyline, di-trifluoroacetate(Example 2.9)

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 90:10:1]; precipitation frommethanol with ether, subsequently freeze drying from water/dioxane.Yield: 68% [TLC: methylene chloride/methanol/glacial acetic acid 80:20:2R_(f)=0.62].

EXAMPLE 6.19

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-lysyl}-batracyline

6.19.a)N-{N^(α),N^(ε)-Bis-[O-(β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl}-batracyline

Educts:

297 mg (1 mmol) of p-aminophenyl β-L-fucoside

66 mg (0.1 mmol) ofN-[lysyl-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracyline,di-trifluoroacetate (Example 2.15)

206 μl of ethyldiisopropylamine

Purification by two precipitations from methanol/methylene chloride(1:1) with ether, washing of the filter residue with ether. Yield: 89%[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.31].

6.19)N-{N^(α),N^(ε)-Bis-[O-(β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-lysyl}-batracyline

515 mg (0.39 mmol) of the compound from Example 6.19.a are dissolved in5 ml of dimethylformamide and 5 ml of piperidine and the solution isstirred at 20° C. for 30 minutes. The batch is concentrated and theresidue is digested with ether. The mixture is filtered with suction andthe filter residue is taken up in dimethylformamide. After precipitationwith ether and rinsing off the filter residue, 335 mg (78%) ofcrystalline target product remain. FAB-MS: m/z=1100=M+1.

EXAMPLE 6.20

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-diaminopropionyl}-batracyline

6.20.a)N-{N^(α),N^(ε)-Bis-1O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-N^(β)-(fluorenyl-9-methoxycarbonyl)-diaminopropionyl}-batracyline

Synthesis analogous to Example 6.19:

Educts:

60 mg (0.22 mmol) of carbohydrate from Example 1.2

100 mg (0.1 1 mmol) ofN-[lysyl-N^(β)-(fluorenyl-9-methoxycarbonyl)-diamino-propionyl]-batracyline,di-trifluoroacetate (Example 2.16)

76 μl of ethyldiisopropylamine

Purification by two precipitations from methanol with ether, washing ofthe filter residue with ether. Yield: 105 mg (73%).

6.20)N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-diaminopropionyl}-batracyline

103 mg (0.079 mmol) of the compound from Example 6.20.a are deblockedwith piperidine analogously to Example 6.19. Purification by flashchromatography (methylene chloride/methanol/ammonia (17%) 15:3:0.3;later in the same system 15:6:0.6). Freeze drying from water/dioxane.Yield: 21%; [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.32].

EXAMPLE 6.21

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-diaminopropionyl}-batracyline,di-sodium salt

This compound was prepared analogously to Example 6.20 via 2 stages,starting from the carbohydrate from Example 1.10 and the peptideconjugate from Example 2.16. Chromatographic purification can be omittedsince by-products can be removed by digestion with methanol and ether.The product is then converted into the di-sodium salt with 2 equivalentsof a 0.1N sodium hydroxide solution. Yield: 66% over 2 stages. [TLC:acetonitrile/water/glacial acetic acid 10:3:1.5 R_(f)=0.3].

EXAMPLE 6.22

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Educt:

60 mg (0.22 mmol) of carbohydrate from Example 1.2

76 mg (0.11 mmol) of N-[lysyl-seryl]-batracyline, di-trifluoroacetate(Example 2.10)

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]; precipitation from methylenechloride/methanol/ether. Yield: 26%. [TLC: acetonitrile/water 10:1R_(f)=0.39].

EXAMPLE 6.23

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracyline

Educt:

60 mg (0.22 mmol) of carbohydrate from Example 1.2

69 mg (0.11 mmol) of N-[lysyl-D-seryl]-batracyline, di-hydrobromide(Example 2.11)

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]; freeze drying fromdioxane/water. Yield: 29%. [TLC: acetonitrile/water 10:1 R_(f)=0.36].

EXAMPLE 6.24

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracyline

Educt:

80 mg (0.3 mmol) of carbohydrate from Example 1.2

53 mg (0.14 mmol) of N-[lysyl]-batracyline (Example 2.17)

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:1:0.1; later in the same system15:2:0.2]; precipitation from methanol/ether. Yield: 26%. [TLC:methylene chloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.22].

EXAMPLE 6.25

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracyline

Educt:

60 mg (0.19 mmol) of carbohydrate from Example 1.10

24 mg (0.063 mmol) of N-[lysyl]-batracyline (Example 2.17)

Purification by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:2:0.2]; later in the same system15:4:0.5; freeze drying from water. Yield: 26%. [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.25].

EXAMPLE 6.26

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracyline

Thiophosgene (33.5 μl, 0.44 mmol) is added to a solution of compound1.31 (79 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml), while stirring.After 10 minutes, the mixture is concentrated in vacuo and the residueis dried under an oil pump vacuum for 1 hour. The isothiocyanateobtained is dissolved in absolute dimethylformamide (10 ml), andcompound 2.17 (37.7 mg, 0.1 mmol) and ethyldiisopropylamine (0.5 ml) areadded. The mixture is stirred at room temperature for 16 hours and thenconcentrated in vacuo and the residue is purified by flashchromatography [methylene chloride/methanol 30:1→20:1→10.1]. Yellowcrystals (56.7 mg, 53%) are obtained; TLC [ethyl acetate/ethanol 2:1]:R_(f)=0.72; melting point=125° C. (decomposition).

EXAMPLE 6.27

N-{N^(α),N^(ε)-Bis-[O-(3-O-methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracyline

Compound 1.42 (75.5 mg, 0.22 mmol) is reacted with peptide conjugate2.17 (37.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 30:1→20:1→10.1] givesyellow crystals (51.1 mg, 44%); TLC [ethanol]: R_(f)=0.80; meltingpoint=176° C. (decomposition).

EXAMPLE 6.28

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate2.17 (37.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (53 mg, 47 %); TLC [methanol]; R_(f)=0.75; meltingpoint=>260° C. (decomposition).

EXAMPLE 6.29

N-{N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracyline

Compound 1.44 (72.2 mg, 0.22 mmol) is reacted with peptide conjugate2.17 (37.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (55.6 mg, 48%); TLC [ethanol]: R_(f)=0.09; meltingpoint=206° C. (decomposition).

EXAMPLE 6.30

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.25 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26. Afterconcentration in vacuo, the residue is taken up in methylenechloride/methanol 1:1 (10 ml) and the product is precipitated byaddition of diethyl ether (15 ml) and washed with a little ice-coldmethanol. Yellow crystals (46 mg, 41%) are obtained; TLC [ethylacetate/ethanol 2:11: R_(f)=0.12; melting point=190-191° C.

EXAMPLE 6.31

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

p-Aminophenyl β-L-fucopyranoside (56.2 mg, 0.22 mmol) is reacted withpeptide conjugate 2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26.Purification by flash chromatography [ethyl acetate→ethanol] andreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergive yellow crystals (73.8 mg, 70%); TLC [ethanol]: R_(f)=0.15; meltingpoint=123° C.

EXAMPLE 6.32

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.31 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.10(69.3 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate→ethyl acetate/ethanol 7:1] gives yellowcrystals (43.8 mg, 38%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.31;melting point=176° C.

EXAMPLE 6.33

N-{N^(α),N^(ε)-Bis-[-(3-O-methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.42 (75.5 mg, 0.22 mmol) is reacted with peptide conjugate2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [ethyl acetate→ethyl acetate/ethanol 3:1] givesyellow crystals (46.2 mg, 37%); TLC [ethyl acetate/ethanol 2:1]:R_(f)=0.12; melting point=161° C.

EXAMPLE 6.34

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26 and the product ispurified. Yellow crystals (39.2 mg, 31%) are obtained; TLC [methanol]:R_(f)=0.77; melting point=213-215° C. (decomposition).

EXAMPLE 6.35

N-{N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.44 (72.2 mg, 0.22 mmol) is reacted with peptide conjugate2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [ethyl acetate/ethanol 2:1] gives yellow crystals(66.1 mg, 53%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.14; meltingpoint=192° C. (decomposition).

EXAMPLE 6.36

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.40 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26 and the product ispurified. Yellow crystals (48.9 mg, 44%) are obtained; TLC [ethylacetate/ethanol 2:1]: R_(f)=0.10; melting point=204° C.

EXAMPLE 6.37

N-{N^(α),N^(ε)-Bis-[O-(2,3-di-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.41 (66 mg, 0.22 mmol) is reacted with peptide conjugate 2.10(69.3 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate→ethyl acetate/ethanol 7:1] gives yellowcrystals (52 mg, 45%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.28;melting point=164-165° C.

EXAMPLE 6.38

N-{N^(α),N^(ε)-Bis-[O-(β-O-(β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracyline

Compound 1.56 (95.4 mg, 0.22 mmol) is reacted with peptide conjugate2.10 (69.3 mg, 0.1 mmol) as described in Example 6.26. Afterconcentration in vacuo, the product is washed thoroughly with hotmethanol (50 ml). Yellow crystals (6.16 mg, 44%) are obtained; TLC[methanol]: R_(f)=0.32; melting point=222° C. (decomposition).

EXAMPLE 6.39

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracyline

p-Aminophenyl β-L-fucopyranoside (56.2 mg, 0.22 mmol) is reacted withpeptide conjugate 2.11 (62.6 mg, 0.1 mmol) as described in Example 6.26.Purification by flash chromatography [ethyl acetate→ethanol] andreprecipitation of the residue from methanol/methylene chloride 1:1 withdiethyl ether give yellow crystals (50.9 mg, 48%); TLC [ethanol]:R_(f)=0.14; melting point=133° C.

EXAMPLE 6.40

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracyline

Compound 1.31 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.11(62.6 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate→ethyl acetate/ethanol 5:1] gives yellowcrystals (53.8 mg, 47%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.38;melting point=145-146° C.

EXAMPLE 6.41

N-{N^(α),N^(ε)-Bis-[O-(3-O-methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracyline

Compound 1.42 (75.5 mg, 0.22 mmol) is reacted with peptide conjugate2.11 (62.6 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [ethyl acetate→ethyl acetate/ethanol 3:1] givesyellow crystals (52.4 mg, 42%); TLC [ethyl acetate/ethanol 2:1]:R_(f)=0.12; melting point=168° C.

EXAMPLE 6.42

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate2.11 (62.6 mg, 0.1 mmol) as described in Example 6.26 and the product ispurified. Yellow crystals (69.2 mg, 55%) are obtained; TLC [methanol]:R_(f)=0.71; melting point=214-216° C. (decomposition).

EXAMPLE 6.43

N-{N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracyline

Compound 1.44 (72.2 mg, 0.22 mmol) is reacted with peptide conjugate2.11 (62.6 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [ethyl acetate/ethanol 2:1] gives yellow crystals(46.4 mg, 38%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.10; meltingpoint=173° C.

EXAMPLE 6.44

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline

p-Aminophenyl β-L-fucopyranoside (56.2 mg, 0.22 mmol) is reacted withpeptide conjugate 2.11 (72.1 mg, 0.1 mmol) as described in Example 6.26.Purification by reprecipitation from methanol/methylene chloride 1:1with diethyl ether gives yellow crystals (69.4 mg, 64%); TLC [ethanol]:R_(f)=0.14; melting point=185-187° C.

EXAMPLE 6.45

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline

Compound 1.31 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.7(72.1 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/acetic acid 200:1→ethyl acetate/ethanol3:1] gives yellow crystals (99.5 mg, 85%); TLC [ethanol]: R_(f)=0.59;melting point=149° C. (decomposition).

EXAMPLE 6.46

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate 2.7(72.1 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (93.4 mg, 73%); melting point=220° C.(decomposition).

EXAMPLE 6.47

N-{N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline

Compound 1.44 (72.2 mg, 0.22 mmol) is reacted with peptide conjugate 2.7(72.1 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/ethanol/acetic acid 400:100:2] givesyellow crystals (69.7 mg, 57%); TLC [ethanol]: R_(f)=0.11; meltingpoint=111° C.

EXAMPLE 6.48

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline,sodium salt

Compound 6.44 (21.7 mg, 20 mmol) is suspended in water (10 ml), and0.05N sodium hydroxide solution is added dropwise to the suspension,while stirring, until a clear solution forms (pH<10). Lyophilization ofthe filtered solution gives a yellow amorphous solid (20.6 mg, 93%).

EXAMPLE 6.49

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline,sodium salt

Compound 6.45 (23.5 mg, 20 mmol) is reacted as described in Example 6.48and the product is worked up. A yellow amorphous solid (23.9 mg, 100%)is obtained.

EXAMPLE 6.50

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline,tri-sodium salt

Compound 6.46 (25.6 mg, 20 μmol) is dissolved in water (10 ml), and0.05N sodium hydroxide solution is added dropwise to the solution, whilestirring, until pH 8 is reached. Lyophilization of the filtered solutiongives a yellow amorphous solid (24 mg, 92%).

EXAMPLE 6.51

N-{N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracyline,sodium salt

Compound 6.47 (24.7 mg, 20 μmol) is reacted as described in Example 6.48and the product is worked up. A yellow amorphous solid (23.0 mg, 92%) isobtained.

EXAMPLE 6.52

N-{N^(α),N^(ε)-Bis-[O-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-glutamyl}-batracyline

p-Aminophenyl β-L-fucopyranoside (56.2 mg, 0.22 mmol) is reacted withpeptide conjugate 2.8 (66.8 mg, 0.1 mmol) as described in Example 6.26.Purification by flash chromatography [ethyl acetate/acetic acid200:1→ethyl acetate/ethanol/acetic acid 10:1:0.1] gives yellow crystals(39.5 mg, 36%); TLC [ethanol]: R_(f)=0.09; melting point=138-139° C.(decomposition).

EXAMPLE 6.53

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-glutamyl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate 2.8(66.8 mg, 0.1 mmol) as described in Example 6.26 and the product ispurified. Yellow crystals (97.4 mg, 75%) are obtained; meltingpoint=180° C. (decomposition).

EXAMPLE 6.54

N-{N^(α),N^(ε)-Bis-O-(3-O-carboxymethyl-β-D-galatopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-glutamyl}-batracyline,tri-sodium salt

Compound 6.53 (25.6 mg, 20 μmol) is reacted as described in Example 6.50and the product is worked up. A yellow amorphous solid (24.3 mg, 92%) isobtained; [α]²⁰=+20.0° (c=0.26/H₂O).

EXAMPLE 6.55

N-N^(α),N^(ε)-Bis-[O-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

p-Aminophenyl β-L-fucopyranoside is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate→ethanol] gives yellow crystals (62.2 mg,60%); TLC [ethanol]: R_(f)=0.12; melting point=176° C.

EXAMPLE 6.56

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-p-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Compound 1.25 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/ethanol 10:1→2:1] gives yellow crystals(56.2 mg, 52%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.22; meltingpoint=197° C.

EXAMPLE 6.57

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Compound 1.31 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/ethanol 10:1→3:1] gives yellow crystals(26.2 mg, 23%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.39; meltingpoint=209° C.

EXAMPLE 6.58

N-{N^(α),N^(ε)-Bis-[O-(3-O-methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Compound 1.42 (75.5 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/ethanol→10:1] gives yellow crystals (22mg, 18%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.06; meltingpoint=194-195° C. (decomposition).

EXAMPLE 6.59

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. After concentration invacuo, the product is washed thoroughly with methanol, methylenechloride and diethyl ether. Yellow crystals (86.8 mg, 71%) are obtained;melting point=230-232° C. (decomposition).

EXAMPLE 6.60

N-{N^(α),N^(ε)-Bis-10-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Compound 1.44 (72.2 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. After concentration invacuo, and washing with methanol, methylene chloride and diethyl ether,yellow crystals (40.9 mg, 35%) are obtained; TLC [ethyl acetate/ethanol2:1]: R_(f)=0.05; melting point=214-216° C. (decomposition).

EXAMPLE 6.61

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-ε-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Compound 1.40 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/ethane 10:1→2:1] gives yellow crystals(72.2 mg, 66%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.18; meltingpoint=175° C.

EXAMPLE 6.62

N-{N^(α),N^(ε)-Bis-[O-(2,3-di-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracyline

Compound 1.41 (66 mg, 0.22 mmol) is reacted with peptide conjugate 2.9(66.2 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [ethyl acetate/ethanol 10:1→3:1] gives yellow crystals(66.6 mg, 60%); TLC [ethyl acetate/ethanol 2:1]: R_(f)=0.41; meltingpoint=173° C.

EXAMPLE 6.63

N-{N^(α),N^(ε)-Bis-[-(β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-threonyl}-batracyline

p-Aminophenyl β-L-fucopyranoside (56.2 mg, 0.22 mmol) is reacted withpeptide conjugate 2.12 (64 mg, 0.1 mmol) as described in Example 6.26.Purification by flash chromatography [ethyl acetate→ethanol] andreprecipitation of the product from methanol/methylene chloride 1:1 withdiethyl ether give yellow crystals (30.5 mg, 28%); TLC [ethanol]:R_(f)=0.10; melting point=172° C.

EXAMPLE 6.64

N-{N^(α),N^(ε)-Bis-[O-(β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.23 (59.7 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 10:1→1:1] gives yellowcrystals (68.3 mg, 64%); TLC [methylene chloride/methanol 1:1]:R_(f)=0.49; melting point=222-224° C. (decomposition).

EXAMPLE 6.65

N-{N^(α),N^(ε)-Bis-[O-(2-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.24 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (69.6 mg, 63%); TLC [ethanol]: R_(f)=0.24; meltingpoint=208° C. (decomposition).

EXAMPLE 6.66

N-{N^(α),N^(ε)-Bis-[O-3-O-methyl-β-D-galactopyranosyl)-4hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.25 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 15:1→10:1→5:1] givesyellow crystals (94.3 mg, 85%); TLC [methylene chloride/methanol 5:1]:R_(f)=0.16; melting point=212° C. (decomposition).

EXAMPLE 6.67

N-{N^(α),N^(ε)-Bis-[O-(4-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.26 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 15:1→10:1→5:1] givesyellow crystals (52.6 mg, 48%); TLC [methylene chloride/methanol]:R_(f)=0.88; melting point=192° C. (decomposition).

EXAMPLE 6.68

N-{N^(α),N^(ε)-Bis-[O-(6-O-methyl-β-D-galactopyranosyl)-4hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.27 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (96.7 mg, 88%); TLC [methylene chloride/methanol5:1]: R_(f)=0.04; melting point=210° C. (decomposition).

EXAMPLE 6.69

N-{N^(α),N^(ε)-Bis-[O-(2,3-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.28 (65.9 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 20:1] gives yellowcrystals (57.4 mg, 51%); TLC [methylene chloride/methanol 5:1]:R_(f)=0.40; melting point=148° C. (decomposition).

EXAMPLE 6.70

N-{N^(α),N^(ε)-Bis-[O-(2,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.29 (65.9 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 20:1→15:1→10:1] givesyellow crystals (74.2 mg, 65%); TLC [methylene chloride/methanol 5:11:R_(f)=0.40; melting point=140° C. (decomposition).

EXAMPLE 6.71

N-{N^(α),N^(ε)-Bis-[O-(2,6-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.30 (65.9 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 20:1→15:1] givesyellow crystals (43.9 mg, 40%); TLC [methylene chloride/methanol 5:1]:R_(f)=0.43; melting point=229° C. (decomposition).

EXAMPLE 6.72

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.31 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 10:1] gives yellow crystals(66.2 mg, 59%); TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.39; melting point=184° C.

EXAMPLE 6.73

N-{N^(α),N^(ε)-Bis-[O-(3,6di-O-methyl-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.32 (65.9 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 15:1→10:13 givesyellow crystals (57.1 mg, 50%); TLC [methylene chloride/methanol 5:1):R_(f)=0.42; melting point=190° C. (decomposition).

EXAMPLE 6.74

N{N^(α),N^(ε)-Bis-[O-(4,6-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.33 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 20:1→10:1→5:1] gives yellowcrystals (47.0 mg, 42%); TLC [methylene chloride/methanol 5:1]:R_(f)=0.39; melting point=169° C.

EXAMPLE 6.75

N-{N^(α),N^(ε)-Bis-[O-(2,3,4-tri-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.34 (69 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 30:1] gives yellow crystals(83.8 mg, 72%); TLC [methylene chloride/methanol 10:1]: R_(f)=0.36;melting point=165° C. (decomposition).

EXAMPLE 6.76

N-{N^(α),N^(ε)-Bis-[(O-(2,3,6-tri-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.35 (69 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (105 mg, 91%); TLC (methylene chloride/methanol5:1]: R_(f)=0.48; melting point=194° C. (decomposition).

EXAMPLE 6.77

N-{N^(α),N^(ε)-Bis-[O-(2,4,6-tri-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.36 (69 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 30:1→10:1] gives yellowcrystals (67 mg, 58%); TLC (methylene chloride/methanol 5:1]:R_(f)=0.54; melting point=228° C. (decomposition).

EXAMPLE 6.78

N-{N^(α),N^(ε)-Bis-[O-(3,4,6-tri-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.37 (69 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (109.3 mg, 94%); TLC [methylene chloride/methanol5:1]: R_(f)=0.52; melting point=180° C. (decomposition).

EXAMPLE 6.79

N-{N^(α),N^(ε)-Bis-[O-(2,3,4,6-tetra-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.38 (69 mg, 0.22 mmol) is reacted with peptide conjugate 2.13(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (99.2 mg, 84%); TLC [methylene chloride/methanol10:1]: R_(f)=0.73; melting point=188° C. (decomposition).

EXAMPLE 6.80

N-{N^(α),N^(ε)-Bis-[O-(3-O-methoxycarbonylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.42 (75.5 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (22 mg, 18%); TLC [methylene chloride/methanol5:1]: R_(f)=0.33; melting point=194-195° C. (decomposition).

EXAMPLE 6.81

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-D-galactopyranosyl)-4hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,di-sodium salt

Compound 1.43 (77.3 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26 and the product ispurified. Yellow crystals (99.7 mg, 81%) are obtained; TLC [methanol]:R_(f)=0.80; melting point=230° C. (decomposition).

EXAMPLE 6.82

N-{N^(α),N^(ε)-Bis-[O-(3-O-carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.44 (72.2 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 7:1] gives yellowcrystals (29.4 mg, 25%); TLC [methylene chloride/methanol 3:1]:R_(f)=0.23; melting point=201-202° C.

EXAMPLE 6.83

N-{N^(α),N^(ε)-Bis-[O-(3,4-O-dideoxy-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.52 (52.6 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 17:1] gives yellowcrystals (38.8 mg, 37%); TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.40; melting point=175° C.

EXAMPLE 6.84

N-{N^(α),N^(ε)-Bis-[O-(α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.39 (59.7 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 10:1→1:1] gives yellowcrystals (52 mg, 48%); TLC [methylene chloride/methanol 1:1]:R_(f)=0.19; melting point 196° C.

EXAMPLE 6.85

N-{N^(α),N^(ε)-Bis-[O-(3,4-di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracyline

Compound 1.31 (79 mg, 0.22 mmol) is reacted with peptide conjugate 2.2(67.7 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 10:1] gives yellow crystals(77.6 mg, 69%); TLC [methylene chloride/methanol/ammonia (25%)15:3:0.2]: R_(f)=0.33; melting point=186° C.

EXAMPLE 6.86

N-{N^(α),N^(ε)-Bis-[O-(4-O-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.56 (95.4 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Afterconcentration in vacuo, the residue is washed thoroughly with hotmethanol (50 ml). Yellow crystals (102.8 mg, 73%) are obtained; TLC[methanol]: R_(f)=0.27; melting point=225-226° C. (decomposition).

EXAMPLE 6.87

N-{N^(α),N^(ε)-Bis-[O-(4-O-(3′-sulphato-β-D-galactopyranosyl)-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline,disodium salt

Compound 1.57 (117.8 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (152.8 mg, 95%); melting point=232° C.(decomposition).

EXAMPLE 6.88

N-{N^(α),N^(ε)-Bis-[O-(4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.58 (98.4 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (118.2 mg, 83%); TLC [methylene chloride/methanol1:1]: R_(f)=0.58; melting point=221° C. (decomposition).

EXAMPLE 6.89

N-{N^(α),N^(ε)-Bis-[O-(2-O-methyl-4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracyline

Compound 1.59 (101.5 mg, 0.22 mmol) is reacted with peptide conjugate2.13 (67.7 mg, 0.1 mmol) as described in Example 6.26. Purification byflash chromatography [methylene chloride/methanol 15:1→10:1→5:1] givesyellow crystals (101.3 mg, 70%); TLC [methylene chloride/methanol 2:1]:R_(f)=0.58; melting point=233° C. (decomposition).

EXAMPLES 7.1-7.13

General Formula

The following glycoconjugates are prepared analogously to Example 4.1.a)starting from other amino acid or peptide conjugates of batracyline orfrom batracyline:

EXAMPLE 7.1

N-{N-(O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracyline

Educts:

carbohydrate from Example 1.2, amino acid conjugate from Example 2.3

Purification by flash chromatography (methylenechloride/methanol/ammonia (17%) 15:1:0.1). Freeze drying fromdioxane/water. Yield: 42% [TLC: methylene chloride/methanol/ammonia(17%) 15:2:0.2): R_(f)=0.31].

EXAMPLE 7.2

N-{N-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracyline,sodium salt

Educts:

carbohydrate from Example 1.10, amino acid conjugate from Example 2.3

Purification by flash chromatography (methylenechloride/methanol/ammonia (17%) 15:3:0.3). Freeze drying fromdioxane/water and subsequent conversion into the sodium salt with 0.1Nsodium hydroxide solution. Yield: 43% [TLC: methylenechloride/methanol/ammonia (17%) 15:4:0.5]: R_(f)=0.14].

EXAMPLE 7.3

N-{N-[O-(β-L-Fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-seryl-D-alanyl]-batracyline

Educts:

p-aminophenyl β-L-fucoside, amino acid conjugate from Example 2.18

Purification by flash chromatography (methylenechloride/methanol/ammonia (17%) 15:3:0.3). Freeze drying fromdioxane/water. Yield: 93% [TLC: methylene chloride/methanol/ammonia(17%) 15:3:0.31: R_(f)=0.28] FAB-MS: m/z=705=M+1.

EXAMPLE 7.4

N-{N-[O-(β-Fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl-D-alanyl}-batracyline

Educts:

p-aminophenyl β-L-fucoside, amino acid conjugate from Example 2.19

Purification by digestion with methanol and precipitation frommethanol/methylene chloride with ether. Yield: 65% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.78].

EXAMPLE 7.5

N-{N-[O-(β-L-Fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-glutamyl-D-alanyl}-batracyline

Educts:

p-aminophenyl β-L-fucoside, amino acid conjugate from Example 2.20

Purification by flash chromatography (methylenechloride/methanol/ammonia (17%) 15:4:0.4; later in the same system15:6:0.6). Freeze drying from dioxane/water. Yield: 92% [TLC: methylenechloride/methanol/ammonia (17%) 15:8:0.8: R_(f)=0.55].

EXAMPLE 7.6

N-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thio-carbonyl]-batracyline

250 mg (1 mmol) of batracyline are dissolved in 50 ml of dioxane and,after addition of 184 μl of thiophosgene, the mixture is stirred at 20°C. for 2 hours. It is concentrated and the residue is digested withether and filtered. The filter residue is dried under a high vacuum for16 hours and then taken up in 30 ml of dimethylformamide. 312 mg (1mmol) of the carbohydrate from Example 1.10 and 500 μl ofethyldiisopropylamine are added and the mixture is treated withultrasound for 4 hours. It is then concentrated and the residue ispurified by flash chromatography (methylene chloride/methanol/ammonia(17%) 15:2:0.2; later in the same system 15:6:0.6). The relevantfractions are concentrated and lyophilized from dioxane/water. Yield:363 mg (60%) [TLC: methylene chloride/methanol/ammonia (17%) 15:6:0.6R_(f)=0.38].

EXAMPLE 7.7

N-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-batracyline

Preparation analogously to Example 7.6 starting from batracyline andcarbohydrate from Example 1.2.

Purification by flash chromatography (methylenechloride/methanol/ammonia (17%) 15:1:0.1); yield: 58% [TLC: methylenechloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.39]. FAB-MS:m/z=561=M+1.

EXAMPLE 7.8

N-{N-[O-(3,4-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-D-alanyl}-batracyline

Compound 1.31 (37.5 mg, 0.11 mmol) is reacted with peptide conjugate 2.3(32 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 30:1] gives yellow crystals(49.3 mg, 75%); TLC [ethyl acetate/ethanol 2.1]: R_(f)=0.81; meltingpoint=185° C. (decomposition).

EXAMPLE 7.9

N-{N-[O-(2,3,4-Tri-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-D-alanyl]-batracyline

Compound 1.34 (34.5 mg, 0.11 mmol) is reacted with peptide conjugate 2.3(32 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 40:1-15:1] gives yellowcrystals (54.9 mg, 81%); TLC [methylene chloride/methanol 20:1]:R_(f)=0.15; melting point=190° C. (decomposition).

EXAMPLE 7.10

N-{N-[O-(3-O-Methoxycarbonylmethyl-β-D-galactopyranosyl)-4hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracyline

Compound 1.42 (37.8 mg, 0.11 mmol) is reacted with peptide conjugate 2.3(32 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 20:1→10:1] gives yellowcrystals (44.3 mg, 63%); TLC [ethanol]: R_(f)=0.85; melting point=195°C. (decomposition).

EXAMPLE 7.11

N-{N-[O-(3-O-Carboxymethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracyline,sodium salt

Compound 1.43 (38.6 mg, 0.11 mmol) is reacted with peptide conjugate 2.3(32 mg, 0.1 mmol) as described in Example 6.26. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl ethergives yellow crystals (63.8 mg, 89%); TLC [ethanol]: R_(f)=0.15; meltingpoint=217° C. (decomposition).

EXAMPLE 7.12

N-{N-[O-(3-O-Carbamoylmethyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracyline

Compound 1.44 (37.8 mg, 0.11 mmol) is reacted with peptide conjugate 2.3(32 mg, 0.1 mmol) as described in Example 6.26. Purification by flashchromatography [methylene chloride/methanol 20:1→10:1→5:1] gives yellowcrystals (20 mg, 29%); TLC [ethanol]; R_(f)=0.15; melting point=184° C.(decomposition).

EXAMPLE 7.13

N-(N-[O-(β-L-Fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracyline

p-Aminophenyl β-L-fucopyranoside (28.1 mg, 0.11 mmol) is reacted withpeptide conjugate 2.3 (32 mg, 0.1 mmol) as described in Example 6.26.Purification by flash chromatography [ethyl acetate/petroleum ether2:1→5:1] gives yellow crystals (49.8 mg, 81%); TLC [ethanol]:R_(f)=0.50; meting point −173° C.

EXAMPLES 8.1-8.12

General Formula

EXAMPLE 8.1

N-{N^(ε)-[2-Chloro-4-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

8.1.a)N-{N^(α)-tert-Butoxycarbonyl]-N^(ε)-[2-chloro-4-[0-3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline

265 mg (0.98 mmol) of p-aminophenyl 3-O-methyl-β-L-fucoside (Example1.2) and 181 mg (0.98 mmol) of cyanuric chloride are taken up in 50 mlof dioxane/water 1:1 and the mixture is cooled to −5° C. and, afteraddition of 83 mg of sodium bicarbonate, stirred at this temperature for15 minutes. 538 mg (0.98 mmol) ofN-[N^(α)-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracyline (Example2.4), dissolved in 14 ml of dimethylformamide, and a further 83 mg ofsodium bicarbonate are then added and the mixture is allowed to come toroom temperature. After stirring at 20° C. for 16 hours, the mixture isconcentrated and the residue is treated with water. The mixture isfiltered with suction and the filter residue is dried under a highvacuum to give 890 mg (96%) of the target product. [TLC: methylenechloride/methanol 10:1 R_(f)=0.26].

8.1)N-{N^(ε)-[2-Chloro-4-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

100 mg (0.11 mmol) of the compound from ample 8.1.a are stirred in amixture of 5 ml of anhydrous trifluoroacetic acid and 5 ml of methylenechloride at 0° C. for 30 minutes. The mixture is concentrated and theresidue is purified by flash chromatography (methylenechloride/methanol/ammonia 17%) 15:1.5:0.15. Subsequent precipitationfrom methanol/ether leads to 41 mg (95%) of the target product [TLC:methylene chloride/methanol/ammonia (17%) 15:2:0.2 R_(f)=0.263 FAB-MS:m/z=829=M+1.

EXAMPLE 8.2

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

8.2.a)N-[N^(α)-[tert-Butoxycarbonyl]-N^(ε)-4-hydroxyethylamino-2-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl]-batracyline

100 mg (0.11 mmol) of the compound from Example 8.1.a are dissolved in 3ml of dioxane. 60 mg of potassium carbonate and 6.5 ml of a 0.1Nsolution of ethanolamine in dioxane are added and the mixture is stirredat 80° C. for 18 hours. It is then concentrated and the residue isstirred with water. The mixture is filtered and the filter residue ispurified by flash chromatography (methylene chloride/methanol/ammonia(17%) 15:1:0.1). After concentration of the relevant fractions anddrying under a high vacuum, 83 mg (80%) of the target compound areobtained [TLC: methylene chloride/methanol/ammonia (17%) 15:2:0.2R_(f)=0.23].

8.2)N-{N^(α)-[4-Hydroxyethylamino-2-10-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

67 mg (0.07 mmol) of the compound from Example 8.2.a are deblockedanalogously to Example 8.1. Purification is carried out by flashchromatography (methylene chloride/methanol/ammonia (17%) 15:2:0.2).Subsequent precipitation from methanol/ether leads to the target productin a 90% yield. FAB-MS: m/z=854=M+1.

The following glycoconjugates are prepared analogously to Example 8.1:

EXAMPLE 8.3

N-{N^(α)-[2-Chloro-4-[-(2-O-methyl-β-L-fucosyl)-4hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.1;

Yield: 76%; FAB-MS: m/z=829=M+1.

EXAMPLE 8.4

N-{N^(ε)-[2-Chloro-4-O-(βL-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

p-aminophenyl β-L-fucoside;

Yield: 36%; FAB-MS: n/z=815=M+1.

[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0, R_(f)=0.44].

EXAMPLE 8.5

N-{N^(ε)-[2-Chloro-4-[O-(3-deoxy-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.6;

Yield: 56%; FAB-MS: m/z=799=M+1;

[TLC: acetonitrile/water/glacial acetic acid 15:1:0.2 R_(f)=0.54].

The following glycoconjugates are prepared analogously to Examples8.1.a, 8.2.a and 8.2 from the batracyline-peptide conjugate in Example2.4 or from the L-alanyl isomer which is to be prepared in acorresponding manner:

EXAMPLE 8.6

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(2-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6yl]-lysyl-D-allyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.1;

Yield: 78%; FAB-MS: m/z=854=M+1;

[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.33].

EXAMPLE 8.7

N{N^(ε)-[4-Hydroxyethylamino-2-[O-(deoxy-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-4-yl-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.4;

Yield: 38%; [TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5R_(f)=0.4].

EXAMPLE 8.8

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(3-deoxy-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.6;

Yield: 77%; FAB-MS: m/z=824=M+1;

[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.37].

EXAMPLE 8.9

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

p-aminophenyl β-L-fucoside;

Yield: 52%; FAB-MS: m/z=840=M+1;

[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.30].

EXAMPLE 8.10

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.2;

Yield: 88%;

[TLC: methylene chloride/methanol/ammonia (17%) 15:3:0.3 R_(f)=0.35].

EXAMPLE 8.11

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(2-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.1;

Yield: 58%;

[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.40].

EXAMPLES 8-12

N-{N^(ε)-[4-Hydroxyethylamino-2-[O-(4-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-alanyl}-batracyline,trifluoroacetate

Educt:

carbohydrate from Example 1.4;

Yield: 66%;

FAB-MS: m/z=854=M+1;

[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.37].

EXAMPLE 9.1

N-[D-Alanyl]-quinolone-a, trifluoroacetate

9.1.a) N-[N-(tert-Butoxycarbonyl)-D-alanyl}-quinolone-a

N-(tert-Butoxycarbonyl)-D-alanine (3.6 g, 19.2 mmol) and2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (5.8 g, 19.2 mmol)are dissolved in 200 ml of dimethylformamide. After the mixture has beenstirred at room temperature for 8 hours, quinolone-a (4 g, 9.6 mmol) and3.3 ml of ethyldiisopropylamine are added and the batch is treated withultrasound for 10 hours. It is concentrated, the residue is taken up inmethylene chloride and the product is precipitated with ether. Afterfiltration, washing with ether and drying under a high vacuum, 4.58 g(81%) of the target product, which is reacted without furtherpurification, are obtained.

9.1) N-[D-Alanyl]-quinolone-a, trifluoroacetate

4.56 g (7.75 mmol) of the compound from Example 9.1.a are dissolved in50 ml of methylene chloride and 50 ml of anhydrous trifluoroacetic acidat 0° C. and the solution is stirred at this temperature for 1 hour. Itis concentrated, the residue is subsequently distilled with methylenechloride and the product is precipitated from methanol with ether. 4.07g (87%) of the crystalline target product are obtained. [TLC:acetonitril/water/glacial acetic acid 5:1:0.2 R_(f)=0.34].

EXAMPLE 9.2

N-[Alanyl]-quinolone-a, trifluoroacetate

The synthesis proceeds in an identical manner to that of the isomer inExample 9.1.

EXAMPLE 9.3

N-[Lysyl-D-alanyl]-quinolone-a, di-trifluoroacetate

9.3.a)N-[N^(α),N^(ε)-Bis-(tert-butoxycarbonyl)-lysyl-D-alanyl]-quinolone-a

341 mg (0.984 mmol) of N^(α),N^(ε)-bis-(tert-butoxycarbonyl)-lysine aredissolved in 10 ml of dimethylformamide, and 200 mg (1.48 mmol) ofN-hydroxybenzotriazole and 227 mg (1.18 mmol) ofN′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride are addedat 0° C. After the mixture has been stirred at 10° C. for 3 hours, 432mg (0.82 mmol) of the compound from Example 9.1 are added and themixture is stirred at 20° C. for a further 2 hours. It is concentratedand the residue is stirred three times with 50 ml of water. The residueis then taken up in methylene chloride and the mixture is dried oversodium sulphate. Precipitation with ether leads to 516 mg (78%) of thetarget product. [TLC: acetonitrile/water 10:1 R_(f)=0.55].

9.3) N-[Lysyl-D-alanyl]-quinolone-a, di-trifluoroacetate

Deblocking of 512 mg (0.63 mmol) of the compound from Example 9.3.aanalogously to Example 9.1. Precipitation from ethyl acetate with ether.Yield: 479 mg (90%); [TLC: acetonitrile/water/glacial acetic acid10:3:1.5 R_(f)=0.3].

EXAMPLE 9.4

N-[Lysyl-alanyl]-quinolone-a, di-trifluoroacetate

The synthesis proceeds in an identical manner to that of the isomer inExample 9.3.

EXAMPLE 9.5

N-[N^(α)-(tert-Butoxycarbonyl)-lysyl-D-alanyl]-quinolone-a

9.5.a)N-[N^(α)-(tert--Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-quinolone-a

1.57 g (3.36 mmol) ofN^(α)-(tert-butoxy-carbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysineare dissolved in 25 ml of dimethylformamide, and 600 mg (5.04 mmol) ofN-hydroxysuccinimide and 820 mg (4.03 mmol) ofN,N′-dicyclohexylcarbodiimide are added at 0° C. After 3 hours, the ureaformed is filtered off, 1.5 g (2.86 mmol) of the compound from Example9.1 are added to the filtrate and the mixture is stirred at roomtemperature for 16 hours. Residual urea is filtered off and the filtrateis purified by flash chromatography (methylene chloride/methanol97.5:2.5; later in the same system 90:10]. The product is thenprecipitated from methylene chloride/methanol 1:1 with ether. Yield: 1.5g (56%) [TLC: methylene chloride/methanol 9:1 R_(f)=0.47].

9.5) N-[N^(α)-(tert-Butoxycarbonyl)-lysyl-D-alanyl]-quinolone-a

Splitting off of Fmoc from Example 9.5.a) with piperidine indimethylformamide. Precipitation of the crude product fromdimethylformamide with ether. Yield: 72% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.43].

EXAMPLE 9.6

N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-quinolone-a,trifluoroacetate

Splitting off of Boc from Example 9.5.a) analogously to Example 9.1.Precipitation of the crude product from methanol/ether. Yield: 80% [TLC:methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.36].

EXAMPLE 9.7

N-[N^(α)-(tert-Butoxycarbonyl)-lysyl-alanyl-quinolone-a

The synthesis proceeds in an identical manner to that of the isomer inExample 9.5.

EXAMPLE 9.8

N-[Lysyl]-quinolone-a, di-trifluoroacetate

9.8.a) N-[N^(α),N^(ε)-Bis-(tert-butoxycarbonyl)-lysyl]-quinolone-a

1317 mg (3.8 mmol) of N^(α),N^(ε)-bis-(tert-butoxycarbonyl)-lysine arelinked with quinolone-a in accordance with the instructions in Example9.1.a. Purification is carried out by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:1:0.1]. 1010 mg (71%) of the targetproduct are obtained.

9.8) N-[Lysyl]-quinolone-a, di-trifluoroacetate

1005 mg (1.347 mmol) of the compound from Example 9.8.a are deblockedanalogously to Example 9.1. After precipitation from methylenechloride/methanol 1:1 with ether, 966 mg (93%) of the crystalline targetproduct are obtained. [TLC: acetonitrile/water/glacial acetic acid10:5:3 R_(f)=0.33].

EXAMPLE 9.9

N-[D-Lysyl]-quinolone-a, di-trifluoroacetate

The synthesis proceeds in an identical manner to that of the isomer inExample 9.8.

EXAMPLE 9.10

N-[N^(α)-(tert-Butoxycarbonyl-lysyl]-quinolone-a

9.10.a)N-[N^(α)-tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-a

1350 mg (2.88 mmol) ofN^(α)-(tert-butoxycarbonyl)-N⁶⁸-(fluorenyl-9-methoxycarbonyl)-lysine arelinked with quinolone-a in accordance with the instructions in Example9.8.a. Purification is carried out by flash chromatography [methylenechloride/methanol/ammonia (17%) 15:1:0.1; later in the same system15:2:0.2]. 1025 mg (82%) of the target product are obtained.

9.10) N-[N^(α)-(tert-Butoxycarbonyl)-lysyl]-quinolone-a

Splitting off of Fmoc from Example 9.10.a analogously to Example 9.5.Two precipitations of the crude product from methanol with ether. Yield:86% [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.48].

EXAMPLE 9.11

N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-a,trifluoroacetate

Splitting off of Boc from Example 9.10.a analogously to Example 9.1. Twoprecipitations of the crude product from methanol/ether. Freeze dryingfrom dioxane/water. Yield: 92% [TLC: methylene chloride/methanol/ammonia(17%) 15:4:0.5 R_(f)=0.44].

EXAMPLES 10.1-10.3

General Formula

EXAMPLE 10.1

N-{N^(ε)-[O(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone-a

10.1.a)N-{N^(α)-(tert-Butoxycarbonyl)-N^(ε)-[O-(3O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone-a

47 μl (0.28 mmol) of thiophosgene are added to 78 mg (0.25 mmol) ofp-aminophenyl 3-O-carboxymethyl-β-L-fucoside (Example 1.10) in 15 ml ofdioxane/water 1:1, while stirring. After the mixture has been stirred at20° C. for 10 minutes, it is concentrated and the residue is dried undera high vacuum for 1 hour. The isothiocyanate obtained is then coupled inabsolute dimethylformamide with 180 mg (0.25 mmol) ofN-[N^(α)-(tert-butoxycarbonyl)-lysyl-D-alanyl)-quinolone-a (Example 9.5)in the presence of 86 μl of ethyldiisopropylamine. After twoprecipitations of the crude product from methylene chloride/ether,subsequent stirring with water and freeze drying from dioxane/water, 210mg (78%) of the target product are obtained. [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.62].

10.1)N-{N^(ε)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}quinolone-a

208 mg (0.193 mmol) of the compound from Example 1.0.a are stirred with10 ml of anhydrous trifluoroacetic acid in 10 ml of methylene chlorideat 0° C. for 1 hour. The mixture is concentrated and the residue issubsequently distilled with 15 ml of methanol and chromatographed withmethylene chloride/methanol/ammonia (17%) 10:10:0.8. After subsequentprecipitation from dimethylformamide with ether, 52 mg (28%) of thetarget product are obtained. [TLC: acetonitrile/water/glacial aceticacid 5:1:0.2 R_(f)=0.53].

The following glycoconjugates are prepared analogously to Example 10.1from the partly protected peptide conjugates in Examples 9.5, 9.7 and9.10:

EXAMPLE 10.2

N-{N^(ε)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-quinolone-a

Educts:

carbohydrate from Example 1.2; peptide conjugate from Example 9.7

Purification of the intermediate stage by several precipitations frommethanol with ether. Flash chromatographic purification of the finalstage with methylene chloride/methanol/ammonia (17%) 15:4:0-5; later inthe same system with 15:8:0.8. Yield: 20% [TLC: methylenechloride/methanol/ammonia (17%) 15:8:0.8 R_(f)=0.15].

EXAMPLE 10.3

N-{N^(ε)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a

Educts:

carbohydrate from Example 1.2; amino acid conjugate from Example 9.10

Purification of the intermediate stage by precipitation from methanolwith ether. Flash chromatography purification of the final stage withmethylene chloride/methanol/ammonia (17%) 15:8:0.8. Yield: 39% [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.33].

EXAMPLES 11.1-11.18

General Formula

EXAMPLE 11.1

N-{N^(α),N^(ε)-Bis[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone-a

50 mg (0.19 mmol) of p-aminophenyl 3-O-methyl-β-L-fucoside (Example 1.2)are first converted into the isothiocyanate in accordance with theinstructions in Example 10.1.a and the product is then coupled in 5 mlof dimethylformamide with 68 mg (0.08 mmol) ofN-[lysyl-D-alanyl]-quinolone-a, di-trifluoroacetate (Example 9.3) in thepresence of 55 μl of ethyldiisopropylamine. The mixture is stirred atroom temperature for 16 hours and concentrated and the residue ispurified by flash chromatography [methylene chloride/methanol/glacialacetic acid 85:15:1.5]. 62 mg (63%) of the target product aresubsequently obtained by precipitation from methanol with ether. [TLC:methylene chloride/methanol/glacial acetic acid 80:20:2 R_(f)=0.5]MS-MALDI: m/z=1242=M+1.

The following glycoconjugates are prepared analogously to Example 11.1from the peptide conjugates in Examples 9.3, 9.4, 9.8 and 9.9:

EXAMPLE 11.2

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-quinolone-a

Educts:

50 mg (0.19 mmol) of carbohydrate from Example 1.2;

0.08 mmol of peptide conjugate from Example 9.4

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 90:10:1] and precipitation frommethanol with ether. Yield: 79% [TLC: acetonitrile/water/glacial aceticacid 5:1:0.2 R_(f)=0.42].

EXAMPLE 11.3

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-quinolone-a

Educts:

52 mg (0.166 mmol) of carbohydrate from Example 1.10;

0.07 mmol of peptide conjugate from Example 9.4

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 80:20:2] and stirring of theresidue with methanol [TLC: acetonitrile/water/glacial acetic acid10:3:1.5 R_(f)=0.62].

EXAMPLE 11.4

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone-a

Educts:

44 mg (0.14 mmol) of carbohydrate from Example 1.10;

0.06 mmol of peptide conjugate 9.3

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 80:20:2] and stirring of theresidue with methanol. Yield: 57%; [TLC: acetonitrile/water/glacialacetic acid 10:3:1.5 R_(f)=0.62].

EXAMPLE 11.5

N-{N^(α),N^(ε)-Bis-[O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-quinolone-a

Educts:

44 mg (0.166 mmol) of carbohydrate from Example 1.21;

0.07 mmol of peptide conjugate from Example 9.4

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 80:20:1] and stirring of theresidue with methanol/ether. Yield: 90 mg (89%); [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.51] MS-ESI:m/z=1212=M+1.

EXAMPLE 11.6

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a

Educts:

70 mg (0.258 mmol) of carbohydrate from Example 1.2;

0.11 mmol of amino acid conjugate from Example 9.8

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 90:10:1] and precipitation frommethylene chloride/methanol 1:1 with ether. Stirring of the residue withwater. Yield: 41%. [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.65].

EXAMPLE 11.7

N-{N^(α),N^(ε)-Bis-[O-(3--methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a

Educts:

50 mg (0.19 mmol) of carbohydrate from Example 1.2;

0.08 mmol of amino acid conjugate from Example 9.9

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 90:10:1] and precipitation frommethylene chloride/methanol 1:1 with ether. Stirring of the residue withwater. Yield: 67%. [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.65] MS-FAB: m/z=1169=M+1.

EXAMPLE 11.8

N-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a,di-sodium salt

Educts:

50 mg (0.16 mmol) of carbohydrate from Example 1.10;

0.07 mmol of amino acid conjugate from Example 9.8

After concentration of the reaction batch, taking up of the residue in10 ml of dimethylformamide and addition of 8 ml of a 0.1N sodiumhydroxide solution, the mixture is stirred at 20° C. for 2 hours. Afterrenewed concentration, the residue is taken up in water and the pH isbrought to 5. The product is lyophilized and digested with methanol andthen with methanol/ether. 68 mg (65%) of the target compound are thusobtained. [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.26].

EXAMPLE 11.9

N-{N^(α,N)^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a,di-sodium salt

Educts:

100 mg (0.32 mmol) of carbohydrate from Example 1.10;

0.13 mmol of amino acid conjugate from Example 12.9

After concentration of the reaction batch, taking up of the residue in10 ml of dimethylformamide and addition of 16 ml of a 0.1N sodiumhydroxide solution, the mixture is stirred at 20° C. for 2 hours. Afterrenewed concentration, the residue is taken up in water and the pH isbrought to 5. The product is lyophilized and digested with methanol andthen with methanol/ether. 160 mg (77%) of the target compound are thusobtained. Melting point: 218-220° C. [TLC: acetonitrile/water/glacialacetic acid 10:3:1.5 R_(f)=0.69].

EXAMPLE 11.10

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-α-L-fucosyl)-4hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a

Educts:

50 mg (0.19 mmol) of carbohydrate from Example 1.3;

0.08 mmol of amino acid conjugate from Example 9.9

Preparation analogously to Example 11.7. Purification by flashchromatography [methylene chloride/methanol/ammonia (17%) 10:10:1] andsubsequent precipitation from methylene chloride/methanol 1:1 withether. Yield: 66%. [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2:R_(f)=0.62].

EXAMPLE 11.11

N-{N^(α),N^(ε)-Bis-O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a

Educts:

50 mg (0.19 mmol) of carbohydrate from Example 1.21;

0.08 mmol of amino acid conjugate from Example 9.9

Preparation analogously to Example 11.7. Yield: 57%. [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.63].

EXAMPLE 11.12

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a,sodium salt

58 mg (0.05 mmol) of the compound from Example 11.7 are suspended inwater and converted into the sodium salt with one equivalent of a 0.1Nsodium hydroxide solution. After freeze drying, 60 mg of the targetcompound are obtained.

EXAMPLE 11.13

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a,sodium salt

Thiophosgene (33.5 ml, 0.44 mmol) is added to a solution of compound1.25 (62.8 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml), while stirring.After 10 minutes, the mixture is concentrated in vacuo and the residueis dried under an oil pump vacuum for 1 hour. The isothiocyanateobtained is dissolved in absolute dimethylformamide (10 ml), andcompound 9.8 (77.4 mg, 0.1 mmol) and ethyldiisopropylamine (0.5 ml) areadded. The mixture is stirred at room temperature for 16 hours and thenconcentrated in vacuo and the residue is purified by flashchromatography (methylene chloride/methanol/ammonia (25%)10:10:1→methanol/ammonia (25%) 20:1]. Yellow crystals are obtained andare suspended in water (10 ml). 0.05N sodium hydroxide solution is addeddropwise to the suspension, while stirring, until a clear solution forms(pH<10). Lyophilization of the filtered solution gives a yellowamorphous solid (39.7 mg, 32%); [α]_(D) ²⁰=+25.8° (c=0.26/H₂O).

EXAMPLE 11.14

N-{N^(α),N^(ε)-Bis-[O-(4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a,sodium salt

Compound 1.58 (98.4 mg, 0.22 mmol) is reacted with peptide conjugate 9.8(77.4 mg, 0.1 mmol) as described in Example 11.13 and the product ispurified. A yellow amorphous solid (62.5 mg, 40%) is obtained; [α]_(D)²⁰=+12.9° (c=0.26/H₂O).

EXAMPLE 11.15

N-{N^(α),N^(ε)-Bis-[O-(2-O-methyl-4-O-(3′-O-Methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a,sodium salt

Compound 1.59 (101.5 mg, 0.22 mmol) is reacted with peptide conjugate9.8 (77.4 mg, 0.1 mmol) as described in Example 11.13. Purification byreprecipitation from methanol/methylene chloride 1:1 with diethyl etherand extraction by boiling with ethanol gives yellow crystals, which areconverted into the sodium salt as described. A yellow amorphous solid(51.1 mg, 32%) is obtained; [α]_(D) ²⁰=+27.9° (c=0.24/H₂O).

EXAMPLE 11.16

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone,sodium salt

Compound 1.25 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate 9.9(77.4 mg, 0.1 mmol) as described in Example 11.13 and the product ispurified. A yellow amorphous solid (77.3 mg, 63%) is obtained; [α]_(D)²⁰=−23.8° (c=0.63/H₂O).

EXAMPLE 11.17

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a,sodium salt

Compound 1.40 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate 9.9(77.4 mg, 0.1 mmol) as described in Example 11.13 and the product ispurified. A yellow amorphous solid (33.6 mg, 27%) is obtained; [α]_(D)²⁰=+0.7° (c=0.28/H₂O).

EXAMPLE 11.18

N-{N^(α),N^(ε)-Bis-[O-(4-O-(3′-O-methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-a,sodium salt

Compound 1.58 (98.4 mg, 0.22 mmol) is reacted with peptide conjugate 9.9(77.4 mg, 0.1 mmol) as described in Example 11.13 and the product ispurified. A yellow amorphous solid (63.0 mg, 41%) is obtained; [α]_(D)²⁰=−21.8° (c=0.22/H₂O).

EXAMPLES 12.1-12.15

General Formula

EXAMPLE 12.1

N-{N′-[O-(3-O-Methyl-β-L-fucosyl)-4hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-a

447 mg (1.66 mmol) of p-aminophenyl 3-O-methyl-β-L-fucoside (Example1.2) are first converted into the isothiocyanate in accordance with theinstructions in Example 10.1.a and the product is then coupled in 40 mlof dimethylformamide with 1 g (1.66 mmol) of N-[D-alanyl]-quinolone-a,trifluoroacetate (Example 9.1) in the presence of 568 μl ofethyldiisopropylamine The mixture is stirred at room temperature for 2hours and concentrated and the residue is purified by severalprecipitations from methylene chloride/methanol 1:1 with ether. Thefilter residue is then stirred twice more with water. 876 mg (66%) ofthe target product are obtained. Melting point: 198° C.; [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.63].

The following glycoconjugates are prepared analogously to Example 12.1from the amino acid conjugates in Examples 9.1 and 9.2:

EXAMPLE 12.2

N-{[N′-[O-(3-O-Methyl-β-L-fucosyl)-4hydroxy-phenylamino-thiocarbonyl]-alanyl}-quinolone-a

Educt:

25 mg (0.092 mmol) of carbohydrate from Example 1.2

Purification by flash chromatography (methylenechloride/methanol/glacial acetic acid 90:10:1], precipitation frommethylene chloride/methanol 1:1 with ether and stirring of the filterresidue with water. Yield: 53 mg (52%). [TLC: acetonitrile/water/glacialacetic acid 5:1:0.2 R_(f)=0.65].

EXAMPLE 12.3

N-{N′-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-a,mono-sodium salt

Educts:

523 mg (1.67 mmol) of carbohydrate from Example 1.10;

840 mg (1.39 mmol) of the compound from Example 12.1

After a reaction time of 6 hours, the mixture is concentrated and theresidue is stirred with water. Flash chromatography [methylenechloride/methanol/ammonia (17%) 15:8:0.8; later in the same system10:1:1] follows freeze drying from dioxane/water. The product is thentaken up in water, one equivalent of a 0.1N sodium hydroxide solution isadded and lyophilization is again carried out. Yield: 525 mg (45%).[TLC: acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.39].

EXAMPLE 12.4

N-{N′-[O-(α-L-Rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-a

Educt:

20 mg (0.076 mmol) of carbohydrate from Example 1.21

Purification by flash chromatography [methylenechloride/methanol/glacial acetic acid 90:10:1] and precipitation frommethanol with ether. Yield: 20 mg (34%). [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.42].

The following glycoconjugates are prepared from partly protectedN-(lysyl)-quinolone-a conjugates and N-(lysyl-D-alanyl)-quinolone-aconjugates:

EXAMPLE 12.5

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a

12.5.a)N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N-^(ε)-[fluorenyl-9-methoxycarbonyl]-lysyl}-quinolone-a

92 mg (0.34 mmol) of p-aminophenyl 3-O-methyl-β-L-fucoside (Example 1.2)are first converted into the isothiocyanate in accordance with theinstructions in Example 10.1.a and the product is then coupled in 20 mlof dimethylformamide with 300 mg (0.34 mmol) ofN-[N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-a,trifluoroacetate (Example 9.11) in the presence of 116 μl ofethyldiisopropylamine. The mixture is stirred at room temperature for 16hours and concentrated and the residue is purified by precipitation frommethylene chloride with ether. The filter residue is then stirredfurther with water and lyophilized from dioxane/water. 290 mg (79%) ofthe target product are obtained. [TLC: acetonitrile/water 10:1R_(f)=0.6].

12.5)N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a

288 mg (0.267 mmol) of the compound of Example 12.5.a are dissolved in20 ml of methylene chloride, and 8 ml of piperidine are added. After themixture has been stirred at 20° C. for 30 minutes, it is concentratedand the residue is precipitated from methylene chloride with ether. Theproduct is purified by flash chromatography [methylenechloride/methanol/ammonia (17%) 10:10:2]. The residue is stirred withether and lyophilized from water. 90 mg (39%) of the target product areobtained. [TLC: methylene chloride/methanol/ammonia (17%) 10:10:5R_(f)=0.4].

The following glycoconjugates are prepared analogously to Examples 12.5from the conjugates in Example 9.11 and 9.6:

EXAMPLE 12.6

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a,di-sodium salt

Educts:

63 mg (0.2 mmol) of carbohydrate from Example 1.10;

158 mg (0.18 mmol) of compound from Example 9.11

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol/ammonia (17%) 15:4:0.5; later in the samesystem 10:10:1] and of the final stage by stirring several times withmethanol and washing of the filter residue with ether. Yield: 44%. Theproduct is then suspended in water, the di-sodium salt is prepared with2 equivalents of a 0.1N sodium hydroxide solution and the solution islyophilized. [TLC: acetonitrile/water/glacial acetic acid 10:3:1.5R_(f)=0.34].

EXAMPLE 12.7

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone-a

Educts:

147 mg (0.47 mmol) of carbohydrate from Example 1.10;

448 mg (0.47 mmol) of compound from Example 9.6

Purification of the intermediate stage by two precipitations frommethylene chloride/methanol 1:1 with ether; stirring of the filterresidue with water (yield: 92%). Purification of the final stage byflash chromatography [methylene chloride/methanol/ammonia (17%)10:10:2]; precipitation from dimethylformamide with ether. Yield: 59%.[TLC: acetonitrile/water/glacial acetic acid 10:3:1.5 R_(f)=0.4].

EXAMPLE 12.8

N-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl4-hydroxy-phenylamino-thio-carbonyl]-lysyl}-quinolone-a,hydrochloride

Educts:

compound 1.25 (62.8 mg, 0.22 mmol);

peptide conjugate 9.11 (180.0 mg, 0.2 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1→7:1→2:1]. Yellow crystals (145.7 mg,67%) are obtained; TLC [methylene chloride/methanol 5:1]: R_(f)=0.48.The fluorenyl-9-methoxycarbonyl group is then split off as described inExample 4.5 and the product is purified. Yellow crystals are obtainedand are suspended in water (10 ml). 0.1N hydrochloric acid is addeddropwise to the suspension, while stirring, until a clear solution forms(pH>3). Lyophilization of the filtered solution gives a yellow amorphoussolid (119.4 mg, 66%); [α]_(D) ²⁰=+33.8° (c=0.28/H₂O).

EXAMPLE 12.9

N-{N^(α)-[O-(3,6-Di-O-methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a,hydrochloride

Educts:

compound 1.32 (65.9 mg, 0.22 mmol);

peptide conjugate 9.11 (180.0 mg, 0.2 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1→7:1→1:1]. Yellow crystals (115.0 mg,52%) are obtained; TLC [methylene chloride/methanol 5:1]: R_(f)=0.44.The fluorenyl-9-methoxycarbonyl group is then split off as described inExample 4.5 and the product is purified. Yellow crystals are obtainedand are suspended in water (10 ml). 0.1N hydrochloric acid is addeddropwise to the suspension, while stirring, until a clear solution forms(pH>3). Lyophilization of the filtered solution gives a yellow amorphoussolid (94.3 mg, 51%); [α]_(D) ²⁰=+44.2° (c=0.34/H₂O).

EXAMPLE 12.10

N-{N^(α)-[O-(3-O-Methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-lysyl}-quinolone-a,hydrochloride

Educt:

compound 1.40 (62.8 mg, 0.22 mmol);

peptide conjugate 9.11 (180.0 mg, 0.2 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol 10:1→5:1→1:1]. Yellow crystals (96.7 mg,44%) are obtained; TLC [methylene chloride/methanol 5:1]: R_(f)=0.47.The fluorenyl-9-methoxycarbonyl group is then split off as described inExample 4.5 and the product is purified. Yellow crystals are obtainedand are suspended in water (10 ml). 0.1N hydrochloric acid is addeddropwise to the suspension, while stirring, until a clear solution forms(pH>3). Lyophilization of the filtered solution gives a yellow amorphoussolid (78.2 mg, 43%); [α]_(D) ²⁰=−157.2° (c=0.30/H₂O).

EXAMPLE 12.11

N-{N^(α)-[O-(4-O-(3′-O-Methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-ahydrochloride

Educts:

compound 1.58 (98.4 mg, 0.22 mmol);

peptide conjugate 9.11 (180.0 mg, 0.2 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol/ammonia (25%)20:10:1→10:10:1→methanol/ammonia (25%) 20:1]. Beige crystals (132.1 mg,53%) are obtained; TLC [methylene chloride/methanol/ammonia (25%)10:10:3]: R_(f)=0.60. The fluorenyl-9-methoxycarbonyl group is thensplit off as described in Example 4.5 and the product is purified.Yellow crystals are obtained and are suspended in water (10 ml). 0.1Nhydrochloric acid is added dropwise to the suspension, while stirring,until a clear solution forms (pH>3). Lyophilization of the filteredsolution gives a yellow amorphous solid (90.0 mg, 42%); [α]_(D)²⁰=+192.2° (c=0.27/H₂O).

The following glycoconjugates are prepared in accordance with theinstructions in Example 12.1 starting from unsubstituted quinolone-a:

EXAMPLE 12.12

N-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4hydroxy-phenylamino-thiocarbonyl-quinolone-a,di-sodium salt

Educts:

78.5 mg (0.25 mmol) of carbohydrate from Example 1.10;

70 mg (0.167 mmol) of quinolone-a

After a reaction time of 6 hours, the mixture is concentrated, theresidue is taken up in dimethylformamide and the mixture is stirred with4 ml of a 0.1N sodium hydroxide solution for 1 hour. Purification byflash chromatography [methylene chloride/methanol/ammonia (17%)15:4:0.5. The pH is brought to 7 with a 0.1N sodium hydroxide solutionand lyophilization is carried out. Yield: 60 mg (44%). [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.42] FAB-MS:m/z=773=M−2Na⁺+3H⁺.

EXAMPLE 12.13

N-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-quinolone-a

Educts:

32 mg (0.12 mmol) of carbohydrate from Example 1.2;

50 mg (0.12 mmol) of quinolone-a

Reaction time of 2 hours; purification by flash chromatography[methylene chloride/methanol/glacial acetic acid 9:10:1]; precipitationfrom methylene chloride/methanol 1:1 with ether. Yield: 59 mg (51%).[TLC: acetonitril/water 10:1 R_(f)=0.43].

EXAMPLE 12.14

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-quinolone-a,hydrochloride

86 mg (0.1 mmol) of the compound from Example 12.5 are taken up in waterand converted into the salt with one equivalent of 0.1N hydrochloricacid. After freeze drying, 88 mg of the target compound are obtained.

EXAMPLE 12.15

N-{N^(α)-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-diamino-propionoyl)-quinolone-a,hydrochloride

The glycoconjugate 12.5 is prepared analogously to Example 12.14 viaseveral stages starting fromN^(α)-(tert-butoxycarbonyl)-N^(β)-(fluorenyl-9-methoxycarbonyl)-L-diaminopropionicacid and quinolone-a [TLC: acetonitrile/water/glacial acetic acid5:1:0.2 R_(f)=0.3].

EXAMPLE 13

General Formula

EXAMPLE 13.1

N-{N′-[O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-b

13.1.a) Quinolone-b:4-amino-7-[(3aRS,4RS,7aSR)-4-amino-1,3,3a,4,7,7a-hexa-hydro-isoindol-2-yl]-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid

170 mg (1.5 mmol) of 1,4-diazabicyclo[2.2.2]octane and 152 mg (1.1 mmol)of (3aRS,4RS,7aSR -4-amino-1,3,3a,4,7,7a-hexahydro-isoindole are addedto 310 mg (I mmol) of5-amino-1-cyclopropyl-6,7-difluoro-1,4-dihydro-8-methoxy4-oxo-3-quinolinecarboxylicacid in a mixture of 4 ml of acetonitrile and 2 ml of dimethylformamideand the mixture is heated under reflux for 1 hour. It is concentrated invacuo, the residue is stirred with about 20 ml of water and the residuewhich has precipitated is filtered off with suction and dried at 100° C.in vacuo.

Yield: 301 mg (70% of theory), Melting point: 237-239° C. (withdecomposition).

13.1.b) N-[D-Alanyl]-quinolone-b, trifluoroacetate

The target compound is prepared analogously to Example 9.1 starting fromcompound 13.1.a and N-(tert-butoxycarbonyl)-D-alanine.

13.1)N-{N′-(O-(3-O-Methyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-b

The target compound is prepared analogously to Example 12.1 startingfrom compound 13.1.b and p-aminophenyl 3O-methyl-β-L-fucoside (Example1.2).

EXAMPLE 14

General Formula

Quinolone-c:8-(2amino-5-methyl-8-azabicyclo[4.3.0]non-3-en-8-yl)-1-methyl-7-fluoro-5-oxo-5H-thiazolo[3,2-a]quinoline-4-carboxylicacid

EXAMPLE 14.1

N-{-N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4hydroxy-phenylamino-thio-carbonyl]-lysyl}-quinolone-c,hydrochloride

14.1.a) N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-c,trifluoroacetate

Educts:

N^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysine

(1.4 g, 3.0 mmol);

quinolone-c (820 mg, 1.9 mmol)

The preparation of the intermediate product is carried out analogouslyto Example 9.1.a. Reprecipitation from ethanol/diethyl ether gives paleyellow crystals (1.37 g, 82%), from which compound 14.1.a is liberatedanalogously to Example 9.1.b. Orange crystals (1.25 g, 74%) areobtained; TLC [methylene chloride/methanol/ammonia (25%) 30:10:1]:R_(f)=0.7; melting point 180° C.

14.a)N-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-c,hydrochloride

Compound 1.25 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate14.1.a (178.4 mg, 0.2 mmol) analogous to Example 12.5. Purification ofthe intermediate stage is carried out by flash chromatography [methylenechloride/methanol/ammonia (25%) 30:6:1→30:10:1]. Pale yellow crystals(97.0 mg, 44%) are obtained; TLC [methylene chloride/methanol/ammonia(25%) 30:10:1]: R_(f)=0.23. The fluorenyl-9-methoxycarbonyl group isthen split off as described and the product is purified. Yellow crystalsare obtained and are suspended in water (10 ml). 0.1N hydrochloric acidis added dropwise to the suspension, while stirring, until a clearsolution forms (pH>3). Lyophilization of the filtered solution is ayellow amorphous solid (75.8 mg, 41%); [α]_(D) ²⁰=+12.5° (c=0.27/H₂O).

The following glycoconjugates are prepared analogously to Example 14.1from peptide conjugate 14.1.a:

EXAMPLE 14.2

N-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-lysyl}-quinolone-c,hydrochloride

Educts:

compound 1.2 (59.5 mg, 0.22 mmol)

peptide conjugate 14.1.a (178.4 mg, 0.2 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol/ammonia (25%) 30:6:1→30:10:1]. Pale yellowcrystals (146.6 mg, 67%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 30:6:1]: R_(f)=0.48. Thefluorenyl-9-methoxycarbonyl group is then split off as described and theproduct is converted into the hydrochloride. A yellow amorphous solid(107.7 mg, 60%) is obtained; [α]_(D) ²⁰=+51.6° (c=0.36/H₂O).

EXAMPLE 14.3

N-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-c,di-sodium salt

The glycoconjugate 14.4 is prepared analogously to Example 12.6 viaseveral stages starting from compound 14.1.a [FAB-MS: m/z=911=M−2Na+3H].

EXAMPLE 14.4

N-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-c,hydrochloride

The conjugate is prepared analogously to the isomer in Example 14.2(FAB-MS: m/z=867=M+H].

EXAMPLE 15

General Formula

Quinolone-d:4-(2-amino-8-azabicyclo[4.3.0]non-4-en-8-yl)-1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylicacid

EXAMPLE 15.1

N-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-d,hydrochloride

15.1.a) N-[N^(ε)-(Fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-d,trifluoroacetate

N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysine(1.4 g, 3.0 mmol) is reacted with quinolone-d, hydrochloride (1.28 mg,2.8 mmol) as described in Example 9.1.a. Reprecipitation from methylenechloride/methanol 1:1 with diethyl ether gives beige crystals (1.97 g,83%), from which compound 15.1.a is liberated analogously to Example9.1.b. Beige crystals (1.7 g, 70%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 28:14:1]: R_(f)=0.60; melting point=215°C.

15.1)N-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-lysyl}-quinolone-d,hydrochloride

Compound 1.25 (62.8 mg, 0.22 mmol) is reacted with peptide conjugate15.1.a (173.2 mg, 0.2 mmol) analogously to Example 12.5. Purification ofthe intermediate stage is carried out by flash chromatography [methylenechloride/methanol/ammonia (25%) 28:14:1→methanol/ammonia (25%) 20:1].Beige crystals (140.8 mg, 65%) are obtained; TLC [methylenechloride/methanol/ammonia (25%) 28:14:1]: R_(f)=0.06. Thefluorenyl-9-methoxycarbonyl group is then split off as described and theproduct is purified. Beige crystals are obtained and are suspended inwater (10 ml). 0.1N hydrochloric acid is added dropwise to thesuspension, while stirring, until a clear solution forms (pH>3).Lyophilization of the filtered solution gives a yellow amorphous solid(102.6 mg, 57%); [α]_(D) ²⁰=−49.0° (c=0.26/H₂O).

The following glycoconjugates are prepared analogously to Example 15.1from peptide conjugate 15.1.a:

EXAMPLE 15.2

N-{N^(α)-[O-(4-O-(3′-O-Methyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-d,hydrochloride

Educts:

compound 1.58 (98.4 mg, 0.22 mmol)

peptide conjugate 15.1.a (173.2 mg, 0.2 mmol)

Purification of the intermediate stage by flash chromatography[methylene chloride/methanol/ammonia (25%)20:10:1→10:10:1→methanol/ammonia (25%) 20:1]. Beige crystals (106.5 mg,43%) are obtained; TLC [methylene chloride/methanol/ammonia (25%)10:10:3]: R_(f)=0.51. The fluorenyl-9-methoxycarbonyl group is thensplit off as described and the product is converted into thehydrochloride. A yellow amorphous solid (82.0 mg, 39%) is obtained;[α]_(D) ²⁰=+22.8° (c=0.29/H₂O).

EXAMPLE 16

Glycoconjugates With Melphalan

General Formula

EXAMPLE 16.1

N-{N′-[O-3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-melphalan

16.1.a) N-tert-Butoxycarbonyl-D-alanyl-melphalan

114 mg (0.6 mmol) of N-tert-butoxycarbonyl-D-alanine are dissolved in 10ml of dimethylformamide, and 138 mg ofN′-(3-dimethylaminopropyl)-N-ethyl-carbodiimide, hydrochloride and1-hydroxy-benzotriazole are added at 0° C. After 10 minutes, 153 mg ofmelphalan are added and the mixture is stirred at room temperature for16 hours. It is concentrated and the residue is partitioned betweenmethylene chloride and water. The organic phase is washed, dried oversodium sulphate and concentrated and the residue is then subjected toflash chromatography with methylene chloride/methanol/ammonia (17%)15:2:0.2→15:4:0.5. 134 mg (56%) of the target compound are obtained[TLC: methylene chloride/methanol/ammonia (17%) 15:4:0.5 R_(f)=0.45].

16.1)N-{N′-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-melphalan

Splitting off of the protective group and coupling with the carbohydrateare carried out as described in Examples 9.1 and 12.1. [TLC:acetonitrile/water 10:1 R_(f)=0.26; FAB-MS: m/z=685=M−H.

EXAMPLE 16.2

N-{N′-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanyl}-melphalan

This compound can be prepared analogously to Example 16.1 via severalstages (TLC: acetonitrile/water 10:1 R_(f)=0.2; FAB-MS: m/z=756=M−H).

EXAMPLES 17

Glycoconjugates With Doxorubicin (Adriamycin)

General Formula

EXAMPLE 17.1

N-{N′-[O-(3--Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanyl}-doxorubicin

17.1.a)N-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanine

160 mg (1 mmol) of alanyl-alanine are taken up in 20 ml of dioxane/water1:1, and 1 ml of Hünig base is added. 1.2 mmol of p-aminophenyl3-O-methyl-β-L-fucoside (Example 1.2) are first converted into theisothiocyanate in accordance with instructions 10.1.a and the product isthen added to the solution of the dipeptide. The mixture is stirred atroom temperature for 16 hours and the residue is purified by flashchromatography (acetonitrile/water 15:1). After concentration of thecorresponding fractions, the product is precipitated frommethanol/ether. Yield: 267 mg (57%).

17.1)N-{N′-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanyl}-doxorubicin

48 mg (0.1 mmol) of the compound from Example 17.1.a are dissolved in 10ml of dimethylformamide, and 23.1 mg ofN′-(3-dimethylaminopropyl)-N-ethyl-carbodiimide, hydrochloride and 21 mgof 1-hydroxy-benzotriazole are added. After 5 minutes, 30 mg ofdoxorubicin and 35 μl of Hünig base are added and the mixture is stirredat room temperature for 30 minutes. It is concentrated and the residueis purified by flash chromatography (methylene chloride/methanol 88:12).The corresponding fractions are concentrated and the residue islyophilized from dioxane/water. 20 mg (40%) of the target compound areobtained. [TLC: methylene chloride/methanol 10:1 R_(f)=0.17; ESI:m/z=997=M+H].

EXAMPLE 17.2

N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fuccopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-alanyl}-doxorubicin

17.2.a)N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-alanine

580 mg (1.31 mmol) of the bis-trifluoroacetate of D-lysyl-alanine arelinked with 2.2 equivalents of the carbohydrate from Example 1.2 in thepresence of 1.3 ml of Hünig base as described in Example 17.1.a.Purification by flash chromatography is carried out withacetonitrile/water 10:1. 446 mg (41%) of the target compound areobtained.

17.2)N-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-alanyl}-doxorubicin

Linking of 59 mg of the compound from Example 17.2.a with 20 mg ofdoxorubicin is carried out as described in Example 17.1. 15 mg of theconjugate are obtained. [TLC: methylene chloride/methanol 85:15R_(f)=0.43; FAB-MS: m/z=1365=M+H].

EXAMPLES 18

Glycoconjugates With Camptothecin

General Formula

EXAMPLE 18.1

20-O-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin

18.1.a) 20-O-(Alanyl)-camptothecin, trifluoroacetate

500 mg (1.44 mmol) of camptothecin are dissolved in 20 ml ofdimethylformamide, and 50 mg of 4-dimethylaminopyridine andN-tert-butoxycarbonyl-alanine N-carboxy-anhydride are then added. After3 hours, a further 775 mg ofN-tert-butoxycarbonyl-alanine-N-carboxy-anhydride are added and thesuspension is treated with ultrasound for 16 hours. The mixture isconcentrated, the crude material is taken up in 50 ml of methylenechloride, and 5 ml of trifluoroacetic acid are added at 0° C. After themixture has been stirred for 30 minutes, it is concentrated again andthe product is purified by flash chromatography (acetonitrile/water20:1). The corresponding fractions are collected and concentrated andthe residue is lyophilized from dioxane/water. 712 mg (93%) of thetarget compound are obtained [FAB-MS: m/z=420=M+H].

18.1.b) 20-O-(Lysyl-alanyl)-camptothecin, bis-trifluoroacetate

The conjugate from Example 18.1.a is linked withN^(α),N^(ε)-bis-(tert-butoxycarbonyl)-lysine in accordance with thestandard instructions and the product is then deblocked. The targetcompound is obtained in a 65% yield.

18.1)20-O-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin

p-Aminophenyl 3-O-methyl-β-L-fucoside (Example 1.2) is linked with theconjugate of Example 1 8.1.b analogously to the instructions in Example11.1. Yield: 40% [TLC: acetonitrile/water 10:1 R_(f)=0.44].

EXAMPLE 18.2

20-O-{N^(α)-[O-(3-O-Carboxymethyl-β-fucopyranosyl)-4hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin

18.2.a)20-O-[N-(Fluorenyl-9-methoxycarbonyl)-lysyl-alanyl]-camptothecin,trifluoroacetate

The conjugate from Example 18.1.a is linked withN^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-lysinein accordance with the standard instructions and the product is thendeblocked on the α-amino function. The target compound is obtained in a24% yield. [TLC: acetonitrile/water 20.1 R_(f)=0.15].

18.2.b)20-O-(N^(α)-[O-(3-O-Carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[fluorenyl-9-methoxycarbonyl]-lysyl-alanyl}-camptothecin

The compound from Example 18.1.a is modified with the carbohydratederivative from Example 1.10 analogously to Example 12.6 and 12.5. Thecrude product can be purified by digestion with water and is thenlyophilized from dioxane/water and employed in the next stage withoutfurther characterization.

18.2)20-O-{N^(α)-[O-(3-O-Carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin

The conjugate 18.2.b is deblocked with piperidine in dimethylformamide.After 30 minutes, the mixture is concentrated and the residue isdigested twice with methylene chloride. It is then taken up indimethylformamide and precipitated with methanol/ether. The product isfiltered off with suction, washed with ether and then lyophilized fromdioxane/water. Yield: 86% [TLC: acetonitrile/water/glacial acetic acid5:1:0.2 R_(f)=0.17].

EXAMPLE 18.3

20-O-{N^(α)-[O-3-O-Carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin,sodium salt

62 mg (0.074 mmol) of the conjugate from Example 18.2 are taken up indioxane/water and converted into the sodium salt with one equivalent ofa 0.1N sodium hydroxide solution. Yield: quantitative [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.17].

The following glycoconjugates of camptothecin are prepared analogouslyto Examples 18.1 and 18.2:

EXAMPLE 18.4

20-O-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-camptothecin

EXAMPLE 18.5

20-O-{N^(α),N^(ε)-Bis-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin

EXAMPLE 18.6

20-O-{N^(α)-[O-3-O-Carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin

EXAMPLE 18.7

20-O-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-lysyl-valinyl}-camptothecin

EXAMPLE 18.8

20-O-{N^(α)-[O-(3-O-Methyl-β-D-galactopyranosyl)-4-hydroxy-phenylamino-thio-carbonyl]-lysyl-alanyl}-camptothecin

EXAMPLE 18.9

20-O-{N^(α)[O-(3-O-Carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin,hydrochloride

Compound 18.6 is converted into the hydrochloride with one equivalent of0.01N hydrochloric acid.

EXAMPLE 18.10

20-O-{N^(α)[O-(3-O-Carboxymethyl-β-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin,hydrochloride

Compound 18.2 is converted into the hydrochloride with one equivalent of0.01N hydrochloric acid.

EXAMPLE 18.11

20-O-{N^(α)[O-(3-O-Carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-phenylalanyl}-camptothecin,hydrochloride

EXAMPLE 18.12

20-O-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin,sodium salt

EXAMPLE 18.13

20-O-{N^(α),N^(ε)-Bis-[O-(3-O-carboxymethyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin,sodium salt

EXAMPLE 18.14

20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin,hydrochloride

We claim:
 1. A compound of the formula (I):  K-Sp-L-AA1-AA2-C  (I)wherein K represents an unsubstituted or regioselectively modifiedmonosaccharide or oligosaccharide moiety of the formula (II):

wherein A represents methyl, hydroxymethyl, carboxyl or an ester oramide derived therefrom, alkoxymethyl, acyloxymethyl orcarboxyalkoxymethyl or an ester or amide derived therefrom; or Arepresents CH₂—B, wherein B represents a monosaccharide oroligosaccharide moiety of the formula (II) linked via the anomericcenter thereof; R₂, R₃ and R₄ independently represent hydrogen,hydroxyl, alkoxy, carboxyalkoxy or an ester or amide derived therefrom,hydroxyalkoxy, aminoalkoxy, acyloxy, carboxyalkylcarbonyloxy, sulphato,phosphato, halogen or another monosaccharide or oligosaccharide moiety(II) modified in the same framework and linked via the anomeric center;or R₃ and R₄ have the meaning given above and R₂ represents amino oracylamino; or one of R₂, R₃ or R₄ has the meaning given above and two ofthe radicals R₂, R₃ and R₄ together represent an epoxide group; Sprepresents optionally substituted arylene or alkylene; L represents aradical of the formula:

wherein R represents chlorine or hydroxyalkylamino; AA1 represents adirect bond or an amino acid radical in the D or L configuration,wherein said amino acid radical is optionally protected with aprotective group or said amino acid radical optionally carries a secondK-Sp-L-group in which K, Sp and L, independent of the K-Sp-L-groupdepicted in formula (I), has the above mentioned meanings; AA2represents a direct bond or an amino acid radical in the D or Lconfiguration, wherein said amino acid radical is optionally protectedwith a protective group or said amino acid radical optionally carries asecond K-Sp-L-group in which K, Sp and L, independent of theK-Sp-L-group depicted in formula (I), has the above mentioned meanings;and C represents the radical of a cytostatic or a derivative thereofthat retains cytostatic activity, which optionally carries an amino orhydroxyl group; or a stereoisomer of said compound, or a salt of saidcompound or stereoisomer.
 2. A compound of the formula (I) according toclaim 1, wherein Sp represents an arylene radical wherein K and L arebound thereto in the ortho-, meta- or para-position relative to oneanother, and said arylene is optionally substituted 1 to 4 times bysubstituents independently selected from the group consisting ofhydrogen, methyl, methoxy, hydroxyl, carboxyl, methoxycarbonyl, cyano,nitro, halogen, sulphonyl and sulphonamide; or Sp represents a linear orbranched alkylene radical; and K, L, AA1, AA2 and C are defined as inclaim 1; or a stereoisomer of said compound, or a salt of said compoundor stereoisomer.
 3. A compound of the formula (I) according to claim 1,wherein C represents the radical of a nucleoside, an endiine antibiotic,a cytotoxic peptide antibiotic, a quinolone- or naphthyridonecarboxylicacid or batracyline, 5-fluorouracil, cytosine arabinoside, methotrexate,etoposide, camptothecin, daunomycin, doxorubicin, taxol, vinblastine,vincristine, dynemycin, calicheamycin, esperamycin, quercetin, suramin,erbstatin, cyclophosphamide, mitomycin C, melphalan, cisplatin,bleomycin, staurosporin or another active compound having anantineoplastic action; and K, Sp, L, AA1 and AA2 are defined as in claim1; or a stereoisomer of said compound, or a salt of said compound orstereoisomer.
 4. A compound of the formula (I) according to claim 1,wherein AA1 represents a direct bond or an amino acid radical derivedfrom lysine, alanine, aspartic acid, glutamic acid, glycine, ornithine,tyrosine, valine or serine in the D or L configuration, wherein saidamino acid radical optionally carries a second K-Sp-L-group: and K, Sp,L, AA1, AA2 and C are defined as in claim 1, or a stereoisomer of saidcompound, or a salt of said compound or stereoisomer.
 5. A compound ofthe formula (I) according to claim 1, wherein AA2 represents a directbond or an amino acid radical derived from lysine, alanine, glycine,ornithine, diaminopropionic acid or serine in the D or L configuration,wherein said amino acid radical optionally carries a secondK-Sp-L-group; and K, Sp, L, AA1 and C are defined as in claim 1; or astereoisomer of said compound, or a salt of said compound orstereoisomer.
 6. A compound of the formula (I) according to claim 1,wherein C represents a radical of the formula (III): T-Q  (III) in whichQ represents a radical of the formula:

wherein R^(a) represents alkyl which has 1 to 4 carbon atoms and isoptionally mono- or disubstituted by halogen or hydroxyl, vinyl,cycloalkyl which has 3 to 6 carbon atoms and is optionally substitutedby 1 or 2 fluorine atoms, bicyclo[1.1.1]pent-1-yl,1,1-dimethylpropargyl, 3-oxetanyl, methoxy, amino, methylamino,dimethylamino or phenyl which is optionally mono- or disubstituted byhalogen, amino or hydroxyl; or R^(a) together with R^(e) forms a bridgeof the structure —*O—CH₂—CH(CH₃)—, —*S—CH₂—CH₂—, —*S—CH₂—CH(CH₃)—,—*CH₂—CH₂—CH(CH₃)— or *O—CH₂—N(R^(f))—, wherein the atom marked with *is bonded to the carbon atom of Y; and wherein R^(f) representshydrogen, methyl or formyl; R^(b) represents hydroxyl, alkoxy having 1to 3 carbon atoms or nitromethyl; R^(c) represents hydrogen or methyl;or R^(c) together with R^(g) forms a bridge of the structure —*O—CH₂—,—*NH—CH₂—, —*N(CH₃)—CH₂—, —*N(C₂H₅)—CH₂—, —*N(C₃H₅)—CH₂— or —*S—CH₂—,wherein the atom marked with * is bonded to the carbon atom of D; R^(d)represents hydrogen, CH₃, CH₂F or ═CH₂; X¹ represents hydrogen, halogenor nitro; X² represents hydrogen, halogen, amino, hydroxyl, methoxy,mercapto, methyl, halogenomethyl or vinyl; Y represents N or C—R^(e);wherein R^(e) represents hydrogen, halogen, CF₃, OCH₃, OCHF₂, CH₃, CN,CH═CH₂ or C≡CH; or R^(e) together with R^(a) forms a bridge of thestructure —*O—CH₂—CH(CH₃)—, —*S—CH₂—CH₂—, —*S—CH₂—CH(CH₃)—,—*CH₂—CH₂—CH(CH₃)— or *O—CH₂—N(R^(f))—, wherein the atom marked with *is bonded to the carbon atom of Y; and wherein R^(f) representshydrogen, methyl or formyl; and D represents N or C—R^(g); wherein R^(g)represents hydrogen, halogen, CF₃, OCH₃, OCHF₂, or CH₃; or R^(g)together with R^(c) forms a bridge of the structure —*O—CH₂—, —*NH—CH₂—,—*N(CH₃)—CH₂—, —*N(C₂H₅)—CH₂—, —*N(C₃H₅)—CH₂— or —*S—CH₂—, wherein theatom marked with * is bonded to the carbon atom of D; n represents 1, 2or 3; and T represents a radical of the formula:

wherein R^(h) represents —N(R^(k))—, —CH₂—O— or —CH₂—N(R^(k))—; whereinR^(k) represents hydrogen or methyl; and R^(i) represents hydrogen,C₁-C₃-alkyl or cyclopropyl; and K, Sp, L, AA1 and AA2 are defined as inclaim 1; or a stereoisomer of said compound, or a salt of said compoundor stereoisomer.
 7. A compound of the formula (I) according to claim 6,in which Q represents a radical of the formula:

wherein R^(a) represents alkyl which has 2 to 4 carbon atoms and isoptionally substituted by 1 fluorine atom, cyclopropyl which isoptionally substituted by 1 fluorine atom, or phenyl which is optionallymono- or disubstituted by fluorine; or R^(a) together with R^(e) forms abridge of the structure —*O—CH₂—CH(CH₃)— or *O—CH₂—N(R^(f))—, whereinthe atom marked with * is bonded to the carbon atom of Y; and whereinR^(f) represents methyl; R^(b) represents hydroxyl or alkoxy having 1 or2 carbon atoms; R^(c) represents hydrogen or methyl; or R^(c) togetherwith R^(g) forms a bridge of the structure of the structure —*O—CH₂—,—*NH—CH₂—, —*N(CH₃)—CH₂— or —*S—CH₂—, wherein the atom marked with * isbonded to the carbon atom of D; X¹ represents fluorine; X² representshydrogen or amino; Y represents N or C—R^(e); wherein R^(e) representshydrogen, fluorine, chlorine, CF₃, OCH₃, OCHF₂ or C≡CH; or R^(e)together with R^(a) forms a bridge of the structure —*O—CH₂—CH(CH₃)— or*O—CH₂—N(R^(f))—, wherein the atom marked with * is bonded to the carbonatom of Y; and wherein R^(f) represents methyl; and D represents N orC—R^(g); wherein R^(g) represents hydrogen, fluorine, chlorine, CF₃,OCH₃ or CH₃; or R^(g) together with R^(c) forms a bridge of thestructure —*O—CH₂—, —*NH—CH₂—, —*N(CH₃)—CH₂— or —*S—CH₂—, wherein theatom marked with * is bonded to the carbon atom of D; and T represents aradical of the formula:

wherein R^(h) represents —NR^(k); wherein R^(k) represents hydrogen ormethyl; and R^(i) represents hydrogen or methyl; or a stereoisomer ofsaid compound, or a salt of said compound or stereoisomer.
 8. Acomposition for treating a mammal afflicted with a tumor comprising aneffective amount therefor of a compound according to any one of claims1-7 and a pharmaceutically acceptable carrier.
 9. A method of treating amammal afflicted with a tumor comprising administering to said mammal aneffective amount therefor of a compound according to any one of claims1-7.